Protein Purification Technical Handbook - Fisher Scientific

Thermo Scientific Pierce ®Protein Purification Technical Handbook

Table of ContentsThermo Scientific Products for Protein PurificationIntroduction 1-2Binding and Elution Buffers for Affinity Purification 3-5BupH Buffer Packs 4-5Solid Supports for Affinity Purification 6-9Columns for Affinity Purification 8-9Covalent Coupling of Affinity Ligands to Chromatography Supports 10-25AminoLink Plus and AminoLink Immobilization Kits and Coupling Resin 14-15UltraLink Biosupport and Pierce CDI Supports 16SulfoLink Kits and SulfoLink Coupling Resin 17UltraLink Iodoacetyl Resin and Micro Peptide Coupling Kit 18Disulfide Reducing Agents 19CarboLink Kit, CarboLink Coupling Resin and UltraLink Hydrazide 20-21PharmaLink Immobilization Kit 22Pierce Maleic Anhydride Plates and Maleimide Activated Plates 23-24Avidin:Biotin Binding 26-35Immobilized Avidin and Streptavidin Products 28Immobilized NeutrAvidin Products 29Immobilized Monomeric Avidin and Kit and Immobilized Iminobiotin and Biotin 30Pierce NeutrAvidin and Streptavidin Coated Plates 32-35Affinity Purification of Antibodies 36-53Protein A and Protein G 38-41Protein A/G and Protein L 42-43IgG Binding and Elution Buffers for Protein A, G, A/G and L 44-45Melon Gel Purification Products 46-47Thiophilic Gel Antibody Purification 48-49IgM Purification, IgA Purification and Immobilized Jacalin 50-51Chicken IgY Purification 52Affinity Purification for Specific Antibodies 53Immunoprecipitation and Co-Immunoprecipitation Assays 54-62Pierce Classic and Seize Immunoprecipitation Kits55-58Pierce Co-Immunoprecipitation Kits 59Pierce HA and c-Myc IP/Co-IP Kits 60Pierce Plus IgG Orientation Kits 61Affinity Based Contaminant Removal 62-65Detergent Removing Gel 63Pierce Blue Albumin Removal Kit 64Detoxi-Gel Endotoxin Removing Gel 65Immobilized E. coli Lysate and Kit 65Additional Affinity Supports 66-75Immobilized Soybean Trypsin Inhibitor 66Immobilized Pepstatin 66Immobilized Heparin 67Immobilized p-Aminophenyl Phosphoryl Choline 67Immobilized D-Galactose 67Immobilized Boronic Acid 67Pierce Cell Surface Protein Isolation Kit 68-69Phosphoprotein Enrichment Kit 70Pierce ConA and WGA Glycoprotein Isolation Kits 71Pierce Ubiquitin Enrichment Kit 72HisPur Cobalt Resin and Spin Columns 73MagnaBind Beads and Supports 74Polystyrene Hydrazide Beads and Polystyrene Beads, Underivatized 75Affinity Supports 76-79Appendices 80

IntroductionActivated affinity support products and kits enable a researcher toimmobilize nearly any type of ligand to purify its binding partner(s).For example, if a peptide antigen is used to immunize animals andproduce antibodies, the same peptide can be immobilized to agel support and used to affinity-purify the specific antibody fromanimal serum. Alternatively, if a specific antibody is availableagainst a particular protein of interest, it can be immobilized toa support and used to affinity-purify the protein from crude celllysate. Purification with respect to nearly any binding interactioncan be made by this approach.Affinity purification products using either immobilized ligandsor activated affinity support chemistries are available for usein several different formats. Most commonly, porous beaded gelsupports are used for gravity-column, spin-column or batch-scalepurification procedures. Coated microplates are available forhigh-throughput screening applications, and magnetic particlesare especially useful for affinity-based cell separation.Affinity PurificationVarious methods are used to enrich or purify a protein of interestfrom other proteins and components in a crude cell lysate orother sample. The most powerful of these methods is affinitypurification, also called affinity chromatography, whereby theprotein of interest is purified by virtue of its specific bindingproperties to an immobilized ligand. We offer a number ofimmobilized protein or ligand products for affinity purificationof antibodies, fusion-tagged proteins, biotinylated proteins andother proteins for which an affinity ligand is available.Affinity purification makes use of specific binding that occursbetween molecules and is used extensively for the isolation ofbiological molecules. A single pass through an affinity columncan achieve a 1,000- to 10,000-fold purification of a ligand from acrude mixture. From a single affinity purification step, it is possibleto isolate a compound in a form pure enough to obtain a singleband upon SDS-PAGE analysis.In affinity purification, a ligand is immobilized to a solid support.Once immobilized, it specifically binds its partner under mild bufferconditions (often physiological conditions such as phosphatebuffered saline). After binding to the partner molecule, the supportis washed with additional buffer to remove unbound componentsof the sample. An elution buffer is added, disrupting the interactionbetween the ligand and its binding partner by pH extremes(low or high), high salt, detergents, chaotrophic agents or theremoval of some factor required for the pair to bind. Oncereleased, the binding partner can be recovered from the supportusing additional elution buffer. The elution buffer can then beexchanged by dialysis or desalting into a more suitable bufferfor storage or downstream analysis.Proteins and other macromolecules of interest can be purifiedfrom crude extracts or other complex mixtures using a varietyof methods. Precipitation is perhaps the simplest method forseparating one type of macromolecule from another. For example,nucleic acids can be precipitated and thereby purified fromundesired molecules in solution using ethanol, and proteins canbe selectively precipitated in the presence of ammonium sulfate.Most purification methods involve some form of chromatographywhereby molecules in solution (mobile phase) are separatedbased on differences in chemical or physical interaction witha stationary material (solid phase). Gel filtration (also calleddesalting, size-exclusion chromatography or SEC) uses a porousgel material to separate molecules based on size; large moleculesare excluded from the internal spaces of the gel material whilesmall molecules enter the resin pores, resulting in a longer paththrough the column. In ion-exchange chromatography, moleculesare separated according to the strength of their overall ionicinteraction with a solid-phase material. By manipulating bufferconditions, molecules of greater or lesser ionic character canbe bound to or dissociated from the solid-phase material.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.1

mlmlmlmlmlIntroductionIn contrast, affinity chromatography or affinity purification makesuse of specific binding interactions between molecules. A particularligand is chemically immobilized or “coupled” to a solid supportso that when a complex mixture is passed over the column, onlythose molecules having specific binding affinity to the ligand arepurified. Affinity purification generally involves the following steps:1. Incubate crude sample with the immobilized ligand supportmaterial to allow the target molecule in the sample to bind tothe immobilized ligand.2. Wash away unbound sample components from solid support.3. Elute (dissociate and recover) the target molecule from theimmobilized ligand by altering the buffer conditions so thatthe binding interaction no longer occurs.Ligands that bind to general classes of proteins (e.g., Protein A forantibodies) or commonly used fusion protein tags (e.g., glutathionefor GST-tagged proteins) are available in pre-immobilized formsready to use for affinity purification. Alternatively, more specializedligands such as specific antibodies or antigens of interest can beimmobilized using one of several activated affinity supports; forexample, a peptide antigen can be immobilized to a support andused to purify antibodies that recognize the peptide.Most commonly, ligands are immobilized or “coupled” directly tosolid support material by formation of covalent chemical bondsbetween particular functional groups on the ligand (e.g., primaryamines, sulfhydryls, carboxylic acids, aldehydes) and reactivegroups on the support. However, other coupling approaches arealso possible. In the Thermo Scientific GST Orientation Kit (Product# 78201), for example, a GST-tagged fusion protein is first boundto an immobilized glutathione support by affinity interaction withthe GST tag and then chemically crosslinked to the support. Theimmobilized GST-tagged fusion protein can then be used to affinitypurifyits binding partner(s). Likewise, Thermo Scientific Seize ® XImmunoprecipitation Kits (e.g., Product # 45215) and ThermoScientific IgG Orientation Kits (e.g., Product # 44990) involvebinding and subsequent crosslinking of an antibody to immobilizedProtein A or G.Historically, researchers have used affinity purification primarilyto purify individual molecules of interest. Increasingly, proteomicsresearch focuses on determination of disease states, celldifferentiation, normal physiological functions and drug discoveryinvolving interaction and expression of multiple molecules ratherthan individual targets. Consequently, the use of affinity methodshas expanded to purification of native molecular complexes andforms the basis for co-immunoprecipitation (co-IP) and “pull-down”assays involving protein:protein interactions.There are a variety of activated affinity supports that allow aresearcher to purify proteins and other biological molecules ofinterest either alone or when present in complexes with theirbinding partners. Many of these supports are discussed in thefollowing pages.TBS5ml5ml5mlAntigen4ml4ml4ml3ml3ml3ml2ml1mlAntigen2ml1mlAntigen2ml1mlAntigenAntigen1. Immobilize the antigen toan appropriate support.2. Quench the unreactedsites and wash.3. Add the antibody solution.5ml5ml5ml4ml4ml4ml4. Bind anti-antigenantibodies.5. Wash off unboundantibodies.6. Elute anti-antigenantibodies.Typical antibody purification using an immobilized antigen column.2For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific Binding and Elution Buffersfor Affinity PurificationCommon elution systems for protein affinity purification.ConditionBufferpH 100 mM glycine•HCl, pH 2.5–3.0100 mM citric acid, pH 3.050–100 mM triethylamine or triethanolamine, pH 11.5150 mM ammonium hydroxide, pH 10.5Ionic strength and/orchaotropic effectsDenaturingOrganicCompetitor3.5–4.0 M magnesium chloride, pH 7.0 in 10 mM Tris5 M lithium chloride in 10 mM phosphate buffer, pH 7.22.5 M sodium iodide, pH 7.50.2–3.0 sodium thiocyanate2–6 M guanidine•HCl2–8 M urea1% deoxycholate1 % SDS10% dioxane50% ethylene glycol, pH 8–11.5 (also chaotropic)> 0.1 M counter ligand or analogBinding and Elution BuffersMost affinity purification procedures involving protein:ligandinteractions use binding buffers, such as phosphate-bufferedsaline (PBS), at physiologic pH and ionic strength. This isespecially true when antibody:antigen or native protein:proteininteractions are the basis for the affinity purification. Once thebinding interaction occurs, the support is washed with additionalbuffer to remove unbound components of the sample.Nonspecific (e.g., simple ionic) binding interactions can beminimized by moderate adjustments to salt concentration or byadding low levels of detergent in the binding and/or wash buffer.Finally, elution buffer is added to break the binding interaction andrelease the target molecule, which is then collected in its purifiedform. Elution buffer can dissociate binding partners by extremesof pH (low or high), high salt (ionic strength), the use of detergentsor chaotropic agents that denature one or both of the molecules,removal of a binding factor, or competition with a counter ligand.In most cases, subsequent dialysis or desalting is required toexchange the purified protein from elution buffer into a moresuitable buffer for storage or downstream analysis. For moreinformation on dialysis or desalting, download or request oura free high-performance dialysis brochure.The most widely used elution buffer for affinity purification ofproteins is 0.1 M glycine•HCl, pH 2.5–3.0. This buffer effectivelydissociates most protein:protein and antibody:antigen bindinginteractions without permanently affecting protein structure.However, some antibodies and proteins are damaged by low pH,so eluted protein fraction(s) should be neutralized immediatelyby collecting the fractions in tubes containing 1/10th volume ofalkaline buffer such as 1 M Tris•HCl, pH 8.5. Other elution buffersfor affinity purification of proteins are listed in the accompanyingtable. In addition, we offer several preformulated binding andelution buffers designed for affinity purification involving antibodies.See pages 4–5 and 44–45 for our pre-formulated binding andelution buffers.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.3

Thermo Scientific Binding and Elution Buffersfor Affinity PurificationBupH Dry BuffersThe most advanced, versatile, time-saving buffer product line available.The ultimate in convenience1. Reach for the sealed foil pack sitting conveniently onyour bench top.2. Open and add to deionized water.3. The fresh buffer is ready to use in practical aliquots sothere’s no waste.The ultimate in versatility• Routine buffers are designed for use in affinity purificationand a variety of other applications.• Using one buffer source maintains consistency and eliminatesvariables within the lab.• Specialized buffers ideally support your work in specificchemistries and methods.The ultimate in integrity• Unlike stored buffers, BupH Buffers are protected fromcontamination.• Carry out applications with confidence in buffer quality.• “Test-assured” with our commitment to world-class, qualitymanagement standards.The ultimate in time savings• The making of specialized and routine buffers is no longertime-consuming.• No component measurement, pH adjustment, quality validation,preparation tracking or refrigeration hassles.• Move forward with your work by eliminating re-tests dueto buffer problems.BupH Borate Buffer PacksIdeal for protein modification procedures that require an alkaline pH.Each pack yields 50 mM borate, pH 8.5 after adding 500 ml ofdeionized water (20 L total).Ordering InformationProduct # DescriptionPkg. Size28384 BupH Borate Buffer Packs 40 packBupH Carbonate-Bicarbonate Buffer PacksIdeal for microplate coating for RIA and EIA techniques.Each pack yields 0.2 M carbonate-bicarbonate buffer, pH 9.4 whendissolved in 500 ml deionized water (20 L total).Ordering InformationProduct # DescriptionPkg. Size28382 BupH Carbonate-Bicarbonate Buffer Packs 40 packBupH Citrate-Carbonate Buffer PacksGreat for use with Thermo Scientific UltraLink ® Supports.Each pack yields 0.6 M sodium citrate, 0.1 M sodium carbonate, pH9 when dissolved in 100 ml of deionized water (1 L total).Ordering InformationProduct # DescriptionPkg. Size28388 BupH Citrate-Carbonate Buffer Packs 10 pack4For more information, or to download product instructions, visit www.thermo.com/pierce

BupH Citrate-MOPS Buffer PacksGreat for use with Thermo Scientific UltraLink Supports.Each pack yields 0.6 M sodium citrate, 0.1 M MOPS buffer, pH 7.5when dissolved in 100 ml of deionized water (1 L total).Ordering InformationProduct # DescriptionPkg. Size28386 BupH Citrate-MOPS Buffer Packs 10 packBupH Phosphate Buffered Saline PacksIdeal for crosslinking and biotinylation.Each pack yields 500 ml of 0.1 M phosphate, 0.15 M NaCl, pH 7.2when dissolved in 500 ml deionized water (20 L total).Ordering InformationProduct # DescriptionPkg. Size28372 BupH Phosphate Buffered Saline Packs 40 packBupH MES Buffered Saline PacksIdeal for use with carbodiimide-coupling chemistries.BupH MES Buffered Saline Packs are designed for use with carbodiimide-couplingchemistries, such as CarboxyLink CouplingResin (Product # 20266) plus EDC. One pack of BupH MES BufferedSaline dissolved in 500 ml of water yields 0.1 M MES (2-[N-Morpholino]ethanesulfonic acid), 0.9% NaCl, pH 4.7.Ordering InformationProduct # DescriptionPkg. Size28390 BupH MES Buffered Saline Packs 10 packBupH Tris Buffered Saline PacksIdeal all-purpose binding buffer.Each pack yields 500 ml of 25 mM Tris, 0.15 M NaCl, pH 7.2 whendissolved in 500 ml deionized water (10 pack makes 5 L total; 40pack makes 20 L total).Ordering InformationProduct # DescriptionPkg. Size28376 BupH Tris Buffered Saline Packs 40 pack28379 BupH Tris Buffered Saline Packs 10 packBupH Modified Dulbecco’s PBS PacksA ready-to-use PBS for immunoassays.Each pack yields 500 ml of 0.008 M sodium phosphate, 0.002 Mpotassium phosphate, 0.14 M sodium chloride and 0.01 Mpotassium chloride, pH 7.4 when dissolved in 500 ml deionizedwater (20 L total).Ordering InformationProduct # DescriptionPkg. Size28374 BupH Modified Dulbecco’s PBS Packs 40 packTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.5

Solid Supports for Affinity PurificationPorous Beaded ResinsPorous beaded supports generally provide the most useful propertiesfor affinity purification of proteins. We offer affinity purificationproducts in two main porous gel support formats: crosslinkedbeaded agarose and Thermo Scientific UltraLink Biosupport.The various features of these two supports are listed in theaccompanying table. Agarose is good for routine applications butcrushes easily, making it suitable for gravity-flow column or smallscalebatch procedures using low-speed centrifugation. UltraLinkBiosupport is incompressible and can be used in high-pressureapplications with a peristaltic pump or other liquid chromatographysystem. In addition, UltraLink Supports display extremely lownonspecific binding characteristics because they are polyacrylamidebased.Both supports perform well in typical gravity-flow and spincolumn purification and immunoprecipitation procedures.Affinity Purification SupportsAffinity purification involves the separation of molecules in solution(mobile phase) based on differences in binding interaction with aligand that is immobilized to a stationary material (solid phase).A support or matrix in affinity purification is any material to whicha biospecific ligand may be covalently attached. Typically, thematerial to be used as an affinity matrix is insoluble in the systemin which the target molecule is found. Usually, but not always,the insoluble matrix is a solid. Hundreds of substances have beendescribed and employed as affinity matrices.Useful affinity supports are those with a high surface area tovolume ratio, chemical groups that are easily modified for covalentattachment of ligands, minimal nonspecific binding properties,good flow characteristics, and mechanical and chemical stability.When choosing an affinity support or matrix for any separation,the most important question to answer is whether a reliablecommercial source exists for the desired matrix material in thequantities required. Fortunately, we offer a wide range of practicaland efficient matrices in volumes ranging from 1 ml to much largerbulk quantities.Magnetic ParticlesWhen a matrix is required for affinity purification of cells within apopulation, we recommend Thermo Scientific MagnaBind Beads.Magnetic affinity separation is a convenient method for isolatingantibodies, antigens, lectins, enzymes, nucleic acids and cellswhile retaining biological activity. Samples containing the moleculeof interest are incubated with beads that are derivatized with anantibody or other binding partner. A rare earth magnet is used topull the MagnaBind Beads out of solution and onto a surface.The buffer can be carefully removed, containing any non-boundmolecules or cells.MagnaBind Beads consist of a silanized surface over an ironoxide core (see Table). The silanized surface has been derivatizedto contain active groups, such as carboxylic acids or primaryamines, or specific affinity molecules such as streptavidin; ProteinA; Protein G; or goat anti-mouse, anti-rabbit or anti-rat IgG. Due tothe nature of the MagnaBind Beads, strong elution conditions arenot recommended with these products. When using MagnaBindBeads to purify certain cells from a population, elution proceduresare not required, as the beads automatically dissociate from thecells within 48 hours due to cell surface turnover. See page 74for a complete listing of MagnaBind Supports.6For more information, or to download product instructions, visit www.thermo.com/pierce

Physical properties of porous gel supports.Support4% Agarose(crosslinked beaded agarose)6% Agarose(crosslinked beaded agarose)Bead 45–165 µm 45–165 µm 50–80 µmThermo Scientific UltraLink Biosupport(co-polymer of crosslinked bis-acrylamide and azlactone)Exclusion Limit 20,000,000 daltons 4,000,000 daltons 2,000,000 daltons (1,000 Å pore size)Durability Crushes under pressure Crushes under pressure Sturdy (> 100 psi, 6.9 bar)*Types of Chromatography Gravity and small spin columns Gravity and small spin columns FPLC Systems, medium pressure, gravity flowCoupling Capacity Medium Medium HighpH range 3–11 3–11 3–11Form Preswollen Preswollen Dry or Preswollen* Note: The indicated maximum pressure of 100 psi refers to the maximum pressuredrop across the gel bed that the support can withstand. It does not necessarily referto the indicated system pressure shown on a liquid chromatography apparatus becausethe system pressure may not actually be measuring the pressure drop across thecolumn. Typical system pressures are usually much higher due to pumping throughsmall I.D. tubing, auto-samplers, detectors, etc. When packed into a 3 mm ID x 14 cmheight glass column, these exclusive supports have been run to approximately 650 psi(system pressure) with no visual compression of the gel or adverse effects onchromatography. These columns can be run at linear flow rates or 85–3,000 cm/hourwith excellent separation characteristics.Characteristics of underivatized Thermo Scientific MagnaBind Beads.CompositionMagnetizationType of MagnetizationSilanized iron oxide25–35 EMU/gSurface Area > 100 m 2 /gSettling RateEffective DensityNumber of BeadsSuperparamagnetic (no magnetic memory)4% in 30 minutes2.5 g/ml1 x 10 8 beads/mgpH Stability Aqueous solution, above pH 4.0Concentration5 mg/mlNote: To establish a microbe-free preparation, MagnaBind Beads can be washed withantibiotic medium or γ-irradiated.MicroplatesPolystyrene microplates are another type of matrix commonly usedfor immobilization of proteins. Proteins passively adsorb to thepolystyrene surface through hydrophobic interactions. Generally,this adsorption of proteins onto the polystyrene surface occursbest in carbonate/bicarbonate buffer at an alkaline pH (9.0–9.5).In addition, polystyrene surfaces can be derivatized with certainchemistries that will allow peptides and other nonprotein moleculesto adhere to the surface to perform affinity assays in the wells ofthe plates.We offer precoated plates to allow researchers an easy-to-use,consistent method for affinity purification or identification ofspecific molecules of interest. The plates offered include thosespecific for fusion proteins (6xHis, GST and GFP), antibodies(Protein A, Protein G, Protein A/G, Protein L, goat anti-mouse andgoat anti-rabbit IgG), biotin (streptavidin and Thermo ScientificNeutrAvidin ® Protein) and those with reactive chemistries (maleicanhydride and maleimide) to allow binding of nonprotein samplesthat do not adsorb to the plastic microplate well surface. Onlyselected microplate products are featured in this handbook.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.7

Thermo Scientific Pierce Columns for Affinity PurificationSpin Cups and ColumnsThermo Scientific Pierce Spin Columns are convenient tools formanipulating small volumes of affinity supports (5–500 µl) forprotein purification. Add the affinity resin and sample to one ofthe columns, use a microcentrifuge to efficiently wash away contaminantsand elute your purified sample without losing any resinin the process. Spin columns allow you to affinity-purifymore protein in less time!Highlights:• Efficient washing of samples means fewer washes are neededto remove contaminating proteins• Efficient elution of samples means more antigen andco-precipitated proteins are recovered• No resin loss means more consistent IP and co-IP results• No need to decant supernatant from IP or co-IP pellet• Spin protocols drastically reduce the time required for IPs and co-IPs• Low protein-binding polypropylene column constructionminimizes nonspecific bindingSpin Cups – Paper FilterPaper filter with collection tubes.Highlights:• Paper filters are resistant to clogging fromcellular debris• Column Volume: 600 µl• Resin Volume: 20–300 µl• Filter Type: Paper, ~10 µm pore size• Cap Type: Collection tube cap fits onto inserted spin cupSpin Cups – Cellulose Acetate FilterCellulose acetate filter with collection tubes.Highlights:• Used in our IP and Co-IP Kits• Column Volume: 800 µl• Resin Volume: 20–400 µl• Filter Type: Cellulose acetate, 0.45 µm pore size• Cap Type: Collection tube cap fits onto inserted spin cupSpin Columns – Screw CapScrew cap with Luer-Lok Adaptors.Highlights:• Luer-Lok Adaptors allow these columns to be usedfor syringe-based purifications• Column Volume: 900 µl• Resin Volume: 20–400 µl• Filter Type: Polyethylene, ~10 µm pore size• Small & large frit options for different sample sizes• Cap Type: O-ring screw top caps; press-in bottom plugsSpin Columns – Snap CapSnap cap with collection tubes.Highlights:• Used in the Cell Surface Protein Isolationand Glycoprotein Isolation Kits• Column Volume: 1,000 µl• Resin Volume: 20–500 µl• Filter Type: Polyethylene, ~30 µm pore size• Cap Type: Snap cap on column (no cap on collection tube);press-on bottom capsMicro-Spin Columns• Column Volume: 400 µl• Resin Volume: 5–100 µl• Filter Type: Polyethylene, ~30 µm pore size• Cap Type: O-ring screw top caps; press-on bottom capsOrdering InformationProduct # Description69700 Spin Cups – Paper FilterIncludes cups and collection tubes69715 Microcentrifuge TubesCollection Tubes for Product # 6970069702 Spin Cups – Cellulose Acetate FilterIncludes cups and collection tubes69720 Microcentrifuge TubesCollection Tubes for Product # 6970269705 Spin Columns – Screw Capwith Luer-Lok AdaptorsIncludes: Spin Columns, Screw Caps and Column PlugsLuer-Lok AdaptorsLarge Frits (6.8 mm diameter 10 µm pore size)Small Frits (2.7 mm diameter 10 µm pore size)Large and Small Frit Tools69725 Spin Columns – Snap Capwith Collection TubesIncludes: Spin Columns and Bottom CapsCollection TubesPkg. Size50/pkg72/pkg50/pkg72/pkgKit25 each5 each25 each25 each1 eachKit50 each100 each89879 Micro-Spin Columns 50/pkgDisposable Plastic ColumnsAutomatic “stop-flow” action provided by porous polyethylenediscs prevents column beds from drying out.Highlights:• Supplied complete with porous polyethylene discs, stoppers andend caps• Compatible with most types of aqueous buffer eluents commonlyused in chromatography• Can be pre-packed and stored until needed8For more information, or to download product instructions, visit www.thermo.com/pierce

Ordering InformationProduct # Description29920 Disposable Polystyrene ColumnsIdeal for packing 0.5–2.0 ml bed volumes.29922 Disposable Polypropylene ColumnsIdeal for packing 1–5 ml bed volumes.29924 Disposable Polypropylene ColumnsIdeal for packing 2–10 ml bed volumes.29923 Disposable Polypropylene FunnelsBuffer reservoirs that fit Product #’s 29920,29922 and 29924.29925 Disposable Column Trial PackIncludes accessories plus two each of Product #’s29920, 29922 and 29924 and one of Product # 29923.Centrifuge ColumnsPkg. Size100/pkg100/pkg100/pkg50/pkgTrial PackEfficiently handle a wide variety of resin volumes for affinitypurification! Thermo Scientific Pierce Centrifuge Columns areconvenient tools for handling 40 µl–10 ml of an affinity purificationsupport. Add the affinity resin to one of the polypropylene columns,remove the twist-off bottom and allow the resin to pack itself.Then add your sample and allow it to bind to the support. Use acentrifuge to efficiently wash away any contaminants and eluteyour purified protein.Pierce Centrifuge Columns allow you to use a spin-column formatin addition to traditional gravity flow to reduce the time required forcolumn washing and elution. This accelerates sample processingtime and makes multiple-sample processing possible. Centrifugecolumns allow you to affinity-purify more protein in less time!Centrifuge columns are made from low protein-bindingpolypropylene for compatibility with protein purification, andthey fit into standard centrifuge tubes for use in any centrifuge.2 ml Centrifuge ColumnsHighlights:• Total Volume: 5 ml (2 ml resin bed, 3 ml reservoir)• Resin Volume: 2 ml• Filter Type: Polyethylene, ~30 µm pore size• Receiver Tube: Fits standard 15 ml conicalcentrifuge tubes• Cap Type: Screw-top cap• Twist-off bottom and press-on cap to reseal5 ml Centrifuge ColumnsHighlights:• Total Volume: 8 ml (5 ml resin bed, 3 ml reservoir)• Resin Volume: 5 ml• Filter Type: Polyethylene, ~30 µm pore size• Receiver Tube: Fits standard 15 ml conicalcentrifuge tubes• Cap Type: Screw-top cap• Twist-off bottom and press-on cap to reseal10 ml Centrifuge ColumnsHighlights:• Total Volume: 22 ml (10 ml resin bed, 12 ml reservoir)• Resin Volume: 10 ml• Filter Type: Polyethylene, ~30 µm pore size• Receiver Tube: Fits standard 50 ml conicalcentrifuge tubes• Cap Type: Screw-top cap• Twist-off bottom and press-on cap to reseal5ml4ml3ml2ml1ml10ml9ml8ml7ml6ml5ml4ml3ml2ml1mlApplications for Centrifuge Columns:• Affinity purification/affinity chromatography• Immunodepletion• Spin desalting0.8 ml Centrifuge ColumnsHighlights:• Total Volume: 800 µl• Resin Volume: 40–400 µl• Filter Type: Polyethylene, ~30 µm pore size• Receiver Tube: Fits standard microcentrifuge tubes(e.g., Product # 69720)• Cap Type: O-ring screw-top cap• Twist-off bottomOrdering InformationProduct # Description89868 Centrifuge Columns, 0.8 ml capacityIncludes: Pierce Centrifuge ColumnsScrew Caps89869 Centrifuge Columns, 0.8 ml capacityIncludes: Pierce Centrifuge ColumnsScrew Caps89896 Centrifuge Columns, 2 ml capacityIncludes: Pierce Centrifuge ColumnsScrew Caps and Tips89897 Centrifuge Columns, 5 ml capacityIncludes: Pierce Centrifuge ColumnsScrew Caps and Tips89898 Centrifuge Columns, 10 ml capacityIncludes: Pierce Centrifuge ColumnsScrew Caps and Tips69707 Column ExtenderFits 89896, 89897 and 89898. Increases columncapacity by 35 mlPkg. SizeKit50 each50 eachKit4 x 50 each4 x 50 eachKit25 each25 eachKit25 each25 eachKit25 each25 each10 eachTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.9

Covalent Coupling of Affinity Ligandsto Chromatography SupportsThe concept of using immobilized affinity ligands to targetbiomolecules has extended beyond chromatographic applications.Affinity ligands are now coupled to latex beads, nanoparticles,macro-beads, membranes, microplates, array surfaces, dipsticksand a host of other devices that facilitate the capture of specificbiomolecules. The application of affinity targeting includespurification, scavenging (or removal of contaminants), catalysis(or modification of target molecules) and a broad range ofanalytical uses to quantify a target molecule in a sample solution.Covalent Immobilization of LigandsAffinity chromatography uses the specific interactions between twomolecules for the purification of a target molecule. In practice, aligand having affinity for a target molecule is covalently attached toan insoluble support and functions as bait for capturing the targetfrom complex solutions.The affinity ligand can be virtually any molecule that can specificallybind the target without displaying significant nonspecific bindingtoward other molecules in the solution. Ligands that have beenused for affinity separations include small organic compounds thatare able to dock into binding sites on proteins, inorganic metals thatform coordination complexes with certain amino acids in proteins,hydrophobic molecules that can bind nonpolar pockets inbiomolecules, proteins with specific binding regions that are able tointeract with other proteins, and antibodies, which can be designedto target any biomolecule through their antigen-binding sites.Designing custom affinity supports that are able to target uniquebiomolecules requires methods to covalently link a ligand to aninsoluble matrix. Regardless of the intended application, thechemical reactions that make ligand attachment possible arewell characterized and facilitate the attachment of biomoleculesthrough their common chemical groups. The types of functionalitiesgenerally used for attachment include easily reactive componentssuch as primary amines, sulfhydryls, aldehydes, carboxylic acids,hydroxyls, phenolic groups and histidinyl residues. Usually, thesolid-phase matrix first is activated with a compound that isreactive toward one or more of these functional groups. Theactivated complex then can form a covalent linkage betweenthe ligand and the support, resulting in ligand immobilization.The type of linkage that is formed between the matrix and theimmobilized ligand affects the performance of the affinity supportin a number of ways. A linkage that allows the coupled ligand toleach from the matrix will result in contamination of the purifiedprotein and shorten the useful life of the affinity support. A linkagethat introduces a charged functional group into the support cancause nonspecific binding by promoting ion-exchange effects.A linkage that alters the structure of the matrix can change theflow and binding characteristics of the support. Cyanogenbromide (CNBr)-activated supports are informative as an exampleof these principles. This popular immobilization method resultsin a linkage that:1. Has a constant leakage of ligand from the matrix thatbecomes a contaminant in the purified preparation.2. Includes a charged isourea group in the linkage,resulting in nonspecific binding.3. Causes extensive crosslinking of the matrix, reducing theability of large molecules to penetrate into the interior ofthe resin.We offer a number of activated affinity supports that are designedto couple ligands of every type via stable, uncharged covalentlinkages that avoid introducing undesirable properties into thesupports. The activation chemistry and protocols have beenoptimized to ensure excellent coupling yields with minimal effortunder a variety of conditions. Each activated support comes withinstructions for use and literature references as examples. Theassociated kits contain all the coupling buffers, wash buffers andcolumns necessary to perform the ligand immobilization andproduce a support ready to perform an affinity separation.10For more information, or to download product instructions, visit www.thermo.com/pierce

Coupling Affinity Ligands through Amine GroupsThe most common functional target for immobilizing proteinmolecules is the amine group, which is present on the vastmajority of proteins because of the abundance of lysine side chainε-amines and N-terminal α-amines. Thermo Scientific AminoLink ®Coupling Resin and AminoLink Plus Coupling Resin are preparedfrom crosslinked agarose supports, and they are designed tocreate a stable linkage between amine groups and the supportmaterial. AminoLink Resins are activated to contain numerousaldehyde groups, which can be used to immobilize amine-containingligands by reductive amination.The immobilization reaction using reductive amination involvesthe formation of an initial Schiff base between the aldehyde andamine groups, which then is reduced to a secondary amine bythe addition of sodium cyanoborohydride. The cyanoborohydridereducing agent used during the coupling process is mild enoughnot to cleave disulfides in most proteins, and it will not reduce thealdehyde reactants – only the Schiff base intermediates. It is bestto avoid stronger reducing agents such as sodium borohydridebecause of the potential for disulfide reduction of the protein andreduction of the aldehydes on the support to hydroxyls, effectivelyquenching the reaction. Depending on the type and amount ofligand present, a coupling reaction using reductive amination canachieve immobilization yields of greater than 85%.Thermo Scientific UltraLink Biosupport binding capacity for various proteins.Capacity Protein Coupling Buffer35.0 mg/ml Myoglobin 0.1 M CHES, 1.0 M sodium citrate, pH 9.021.5 mg/ml Penicillin Acylase 0.1 M sodium phosphate, 1.1 M sodiumsulfate, pH 7.420.9 mg/ml α-chymotrypsin 0.1 M borate, 1.5 sodium sulfate, pH 9.035.5 mg/ml BSA 0.1 M borate, 1.5 sodium sulfate, pH 9.029.8 mg/ml Lysozyme 0.1 M borate, 1.0 sodium sulfate, pH 9.021.0 mg/ml Human IgG 0.1 M borate, 1.5 sodium sulfate, pH 9.0A third option for immobilizing amine-containing affinity ligandsis the use of carbonyl diimidazole (CDI) to activate hydroxyls onagarose supports to form reactive imidazole carbamates. Thisreactive group is formed on the support in organic solvent andstored as a suspension in acetone to prevent hydrolysis. Reactionof the support in an aqueous coupling buffer with primaryamine-containing ligands causes loss of the imidazole groupsand formation of carbamate linkages. The coupling processoccurs at basic pH (8.5–10), but it is a slower reaction withproteins than reductive amination or azlactone coupling. ThermoScientific Pierce CDI Supports are available with the CDI-activatedgroup, and they are particularly adept at immobilizing peptides andsmall organic molecules. The reaction also can be done inorganic solvent to permit coupling of water-insoluble ligands.OHH 2 N–RNaCNBH 3NRHNHO NH 2 N—RO NRAldehyde Group onThermo ScientificAminoLink Coupling ResinAffinity Ligand Coupledvia Secondary Amine BondAnother amine-reactive strategy that can be used for immobilizationis the azlactone ring present in UltraLink Biosupport. A primaryamine will react with an azlactone group in a ring-openingprocess that produces an amide bond at the end of a five-atomspacer. The group is spontaneously reactive with amines,requiring no additives or catalysts to drive the coupling process.The UltraLink Biosupport is supplied dry to ensure stability of theazlactone groups until use. Adding a quantity of the support to asample containing a protein or other amine-containing moleculecauses immobilization to occur within about one hour. For proteinimmobilization at high yield, it is recommended that the couplingbuffer contain a lyotropic salt, which functions to drive the proteinmolecules toward the bead surface. This brings the hydrophilicamines close enough to the azlactone rings to react quickly. Thesimple nature of coupling affinity ligands to the UltraLinkBiosupport along with its inherently low nonspecific binding makesit one of the best choices for immobilization.CH 3 ONHH 2 N—RNCH 3N ROHOOOReactive Imidazole Carbamateon Thermo Scientific PierceCDI SupportsOAffinity Ligand Coupledvia Carbamate BondAzlactone Group onThermo ScientificUltraLink BiosupportAffinity Ligand Coupledvia Amide BondTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.11

Covalent Coupling of Affinity Ligandsto Chromatography SupportsCoupling Affinity Ligands through Sulfhydryl GroupsIt is often advantageous to immobilize affinity ligands throughfunctional groups other than just amines. In particular, the thiolgroup can be used to direct coupling reactions away from activecenters or binding sites on certain protein molecules. Becauseamines occur at many positions on a protein’s surface, it is usuallydifficult to predict where an amine-targeted coupling reaction willoccur. However, if sulfhydryl groups that typically are present infewer numbers are targeted for immobilization, then coupling canbe done at discrete sites in a protein or peptide. Thiol groups(sulfhydryls) may be indigenous within a protein molecule or theycan be added through the reduction of disulfides or through theuse of various thiolation reagents. Sulfhydryls also can be addedto peptide affinity ligands at the time of peptide synthesis byadding a cysteine residue at one end of the molecule. This ensuresthat every peptide molecule will be oriented on the support in thesame way after immobilization.Thermo Scientific SulfoLink ® Coupling Resin is designed toefficiently react with thiol-containing molecules and immobilizethem through a thioether linkage. The support contains an iodoacetylgroup at the end of a long spacer arm, which reacts withsulfhydryls through displacement of the iodine. Optimal conditionsfor the reaction are an aqueous environment at slightly basic pH,wherein amines are not very reactive toward the iodoacetylfunction, but thiols are highly reactive due to their increasednucleophilicity. The thioether bond that is formed provides astable linkage to any sulfhydryl-containing molecule.Reactive Iodoacetyl Groupon Thermo ScientificSulfoLink SupportsAffinity LigandCoupled viaThioether BondCoupling Affinity Ligands through Carbonyl GroupsMost biological molecules do not contain carbonyl ketones oraldehydes in their native state. However, it might be useful tocreate such groups on proteins to form a site for immobilization thatdirects covalent coupling away from active centers or binding sites.Glycoconjugates, such as glycoproteins or glycolipids, usuallycontain sugar residues that have hydroxyls on adjacent carbonatoms, which can be periodate-oxidized to create aldehydes.Controlled oxidation using 1 mM sodium meta-periodate at 0°C willselectively oxidize sialic acid groups to form an aldehyde functionalityon each sugar. Using higher concentrations of periodate (10 mM)at room temperature will result in oxidation of other sugar diols tocreate additional formyl groups. Aldehydes on the carbohydrateportion of glycoconjugates can be covalently linked withaffinity supports through an immobilized hydrazide, hydrazine oramine group by Schiff base formation or reductive amination.HSRHNHNOOISRThermo Scientific CarboLink Coupling Resin contains long spacerarms that terminate in hydrazide groups. Reaction of the hydrazideswith aldehydes forms hydrazone linkages, which are a form ofSchiff base displaying better stability than those formed betweenan amine and an aldehyde. The CarboLink Resin can be used toimmobilize glycoproteins, such as antibodies, after periodateoxidation of the carbohydrate. Coupling antibodies in this mannerspecifically targets the heavy chains in the Fc portion of themolecule. Since this is away from the antigen-binding sites at theend of the Fv regions, immobilization using this route often resultsin the best retention of antigen-binding activity.Reactive Hydrazide Groupon Thermo ScientificCarboLink SupportsAffinity LigandCoupled viaHydrazone BondOONHNHNH 2The CarboLink Resin also can be used to couple carbohydrates andsugars through their reducing ends. Aldehyde- or ketone-containingsugars will react with the immobilized hydrazide groups withoutoxidation of other sugar hydroxyls. However, this reaction may bedramatically slower than coupling with oxidized sugars becausethese native aldehydes or ketones are usually tied up in acetal orketal ring structures. These rings can open in aqueous solution toreveal the aldehyde or ketone, but the open structure is presentonly a small percentage of the time. Thus, the reducing ends ofsugars have decreased reactivity toward an immobilized hydrazide,sometimes requiring days of reaction time to obtain acceptableimmobilization yields.Although the hydrazone bond created between the immobilizedhydrazide and an aldehyde is much more stable than amine-aldehydeSchiff bases, to obtain a leach-resistant linkage it is recommendedthat the Schiff base be reduced with sodium cyanoborohydride.This is especially true if a ligand is coupled that has only a singlepoint of attachment to the support. Reduction of the hydrazonecreates a stable bond that will perform well in affinitychromatography applications.Affinity LigandCoupled viaHydrazone BondStable LigandLinkageROONaCNBH 3OHNHNHNHNNRRR12For more information, or to download product instructions, visit www.thermo.com/pierce

Coupling Affinity Ligands through Carboxyl GroupsCoupling Affinity Ligands throughReactive HydrogensHON NH 2 RThe carboxyl group is a frequent constituent of many biologicalmolecules. Particularly, proteins and peptides typically containnumerous carboxylic acids due to the presence of glutamic acid,aspartic acid and the C-terminal α-carboxylate group. Carboxylicacids may be used to immobilize biological molecules through theuse of a carbodiimide-mediated reaction. Although no activatedsupport contains a reactive group that is spontaneously reactivewith carboxylates, chromatography supports containing amines(or hydrazides) may be used to form amide bonds with carboxylates.Molecules containing carboxylates may be activated to reactwith an immobilized amine (or hydrazide) through reaction with thewater-soluble carbodiimide EDC.EDC reacts with carboxylates to form an intermediate ester that isreactive with nucleophiles such as primary amines. The reactiontakes place efficiently between about pH 4.5 and pH 7.5, and it iscomplete within two to four hours, depending on the temperature.The intermediate ester is subject to hydrolysis; therefore, it isbeneficial if the amine-containing ligand to be immobilized isincluded in the reaction medium upon addition of EDC, so it canreact immediately with the ester as it forms.Thermo Scientific CarboxyLink Coupling Resin or the UltraLinkDADPA Resin may be used to immobilize carboxylate-containingligands by EDC. CarboxyLink Resin contains a nine atom spacer armand UltraLink DADPA contains a 12-atom spacer arm to minimizesteric hindrance.HNImmobilized DADPA onThermo Scientific CarboxyLink Resin+OHCarboxylate-ContainingAffinity LigandFor molecules containing no easily reactive functional groups,immobilization may be difficult or even impossible using currenttechnologies. Certain drugs, steroids, dyes and other organicmolecules often have structures that contain no available “handles”for convenient immobilization. In other cases, functional groupsthat may be present on a molecule have low reactivity or aresterically hindered, prohibiting efficient coupling. Often, thesecompounds that are difficult to immobilize will have certain active(or replaceable) hydrogens that can be condensed with formaldehydeand an amine in the Mannich reaction. Certain hydrogens inketones, esters, phenols, acetylenes, α-picolines, quinaldines andother compounds can be aminoalkylated using this reaction.Formally, the Mannich reaction consists of the condensation offormaldehyde (or another aldehyde) with ammonia and anothercompound containing an active hydrogen. Instead of usingammonia, this reaction can be done with primary or secondaryamines or even with amides.Use of the Mannich reaction for the preparation of affinity supportsoffers some unique advantages beyond that of its effective use toimmobilize compounds that are difficult to couple. For instance,polymerization is often a problem when using the Mannich reactionfor solution-phase chemistries, especially when multiple reactivehydrogens are present on a molecule. When one of the reactivespecies is immobilized, the reaction is more controlled and undesirableside reactions are inhibited. Compounds with phenolicresidues (often found in drugs) can be coupled without difficulty.In addition, the Mannich reaction is a superior alternative to theseldom-used diazonium coupling method. Both the diazonium groupand the resultant diazo linkage are unstable. In contrast, immobilizationusing Mannich condensations result in very stable covalentbonds suitable for the most critical affinity separations.HNEDCHNImmobilized Ligand viaAmide Bond FormationHNORWe have developed the Thermo Scientific PharmaLink ImmobilizationKit, which is based on the principles of the Mannich reaction.The PharmaLink Resin included in the kit is immobilized diaminodipropylamine(DADPA), which is the source of the primary aminefor the Mannich reaction. The kits also include coupling buffer,coupling reagent, wash buffer and accessories.OHHOHNHOEstradiol-17β+HN NH 2Immobilized DADPA onThermo Scientific PharmaLink ResinHOH + , 37˚CFormaldehydeHHNHNHNImmobilized Ligand viaAminoalkyl Bond FormationTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.HO13

Thermo Scientific Products for Covalent Couplingof Affinity Ligands to Chromatography SupportsAminoLink Plus Immobilization Kitsand Coupling ResinThe simplest and surest method for making an affinity purificationresin with antibodies or other proteins.Thermo Scientific AminoLink Plus Coupling Resin andImmobilization Kits use activated beaded agarose and a robustcoupling chemistry to immobilize proteins and other ligands throughprimary amines (–NH 2 ) to the resin. Once an antibody or otherligand is immobilized, the prepared affinity resin can be used for avariety of purification methods involving batch or column chromatography.The resin and linkage are stable in binding and elutionconditions typically used in affinity chromatography, enabling preparedresin to be used for at least 10 rounds of affinity purification.The AminoLink Plus Coupling Reaction involves spontaneousformation of Schiff base bonds between aldehydes (on the support)and amines (on the ligand) and their subsequent stabilization byincubation with a mild reductant (sodium cyanoborohydride; seemore detailed reaction scheme on next page). The entire couplingreaction, called reductive amination, occurs in four to six hoursin simple non-amine buffers such as PBS. Coupling efficiency withantibodies and typical proteins is generally greater than 85%,resulting in 1 to 20 mg of immobilized protein per milliliter ofagarose resin.Highlights:• AminoLink Plus Coupling Resin – aldehyde-activated crosslinked4% beaded agarose• Ideal for antibodies and other proteins – immobilize moleculesvia primary amines (–NH 2 )• Flexible coupling conditions – efficient (> 85%) coupling over awide range of pH (4–10) and buffer conditions (PBS or othernon-amine buffer with or without organic solvent); regular(PBS, pH 7.2) and enhanced (borate, pH 10) coupling protocolsprovided• Stable, permanent immobilization – coupling reaction results instable, leak-resistant secondary amine bond between resinand ligand• Better than immobilization to CNBr-activated agarose – bond ismore stable and uncharged, resulting in less nonspecific bindingin affinity purification procedures• Versatile and reusable – prepared affinity resin is adaptable tocolumn and batch affinity techniques and the resin is reusablefor typical applications based on protein binding interactions• Convenient kits and product sizes – choose one- or five-columnkit containing complete sets of buffers, reagents and versatilespin/drip columns, or select bulk resin. Bulk quantities areavailable for manufacturing applicationsEfficient immobilization of antibodies and other proteins. Percent couplingefficiency of different proteins to 2 ml of Thermo Scientific AminoLink PlusCoupling Resin using the pH 7.2 coupling protocol.ProteinProtein Applied(mg/ml)Protein Coupled(mg/ml)Percent CoupledProtein G 4.6 4.0 83Mouse IgG 4.7 4.5 96Rat IgG 4.7 4.4 93Goat IgG 0.9 0.8 84Human IgG 4.8 4.6 97Human IgM 0.9 0.8 93AgaroseBeadOCHThermo ScientificAminoLink Plus Resin(Aldehyde Activated)+H 2 NH 2 NNH 2NH 2Antibody withPrimary AminesYThermo Scientific AminoLink Support immobilization chemistry.ReferencesBeall, A., et al. (1999). J. Biol. Chem. 274(16), 11344–11351.Nakasato, Y.R., et al. (1999). Clin. Chem. 45, 2150–2157.Allan, B.B., et al. (2000). Science 289, 444–448.Lu, R., et al. (2000). J. Neurochem. 74, 320–326Ordering InformationYYYYBeadYYCovalentlyImmobilized AntibodyProduct # Description Pkg. Size44894 AminoLink Plus Immobilization KitIncludes: AminoLink Plus ColumnsNeutral pH Coupling Buffer (pH 7.2)Enhanced Coupling Buffer (pH 10)Quenching BufferWash SolutionSodium Cyanoborohydride SolutionColumn Accessories20394 AminoLink Plus Immobilization Trial KitIncludes: AminoLink Plus Column Reagents and Buffers20475 AminoLink Plus Micro Immobilization KitSufficient reagents for 10 coupling reactionsusing 25–100 µg of protein and 20 affinity purifications.Includes: AminoLink Plus Spin Columns,each containing 400 µl of 25% slurryPhosphate Buffered SalineQuenching BufferSodium Cyanoborohydride SolutionWash SolutionElution BufferMicrocentrifuge Collection TubesKit5 x 2 ml500 ml500 ml60 ml240 ml0.5 mlTrial Kit1 x 2 mlKit10 each1 pack60 ml0.5 ml25 ml50 ml200 each20501 AminoLink Plus Coupling Resin 10 ml14For more information, or to download product instructions, visit www.thermo.com/pierce

YYAminoLink Immobilization Kitsand Coupling ResinLinks with primary amines (lysine residues and N-terminus)on proteins, peptides, antigens or antibodies.Thermo Scientific AminoLink Coupling Resin is crosslinked4% beaded agarose that has been activated with aldehydegroups. Proteins and other molecules with primary amines canbe covalently attached (immobilized) to AminoLink Resin to makechromatography columns for use in affinity purification. Thealdehyde groups form stable secondary amine bonds with primaryamines such as exist in the side chain of lysine (K) residues, whichare generally abundant and readily accessible in proteins. Once aprotein is immobilized, the prepared affinity resin can be used for avariety of batch and column affinity purification methods involvingbinding interactions with the immobilized protein. The resin andlinkage are stable in most binding and elution conditions typicallyused in affinity chromatography, enabling prepared resin to beused for multiple rounds of affinity purification procedures.Highlights:• AminoLink Coupling Resin – aldehyde-activated crosslinked4% beaded agarose• Ideal for antibodies and other proteins – immobilize moleculesvia primary amines (–NH 2 )• Flexible coupling conditions – efficient (> 85%) coupling over awide range of pH (4–10) and buffer conditions (PBS or othernon-amine buffer with or without organic solvent)• Stable, permanent immobilization – coupling reaction results instable, leak-resistant secondary amine bond between resinand ligand• Better than immobilization to CNBr-activated agarose – bond ismore stable and uncharged, resulting in less nonspecific bindingin affinity purification procedures• Versatile and reusable – prepared affinity resin is adaptable tocolumn and batch affinity techniques and the resin is reusablefor typical applications based on protein binding interactionsEfficient immobilization at a variety of pH values. The effect of couplingbuffer pH on percent coupling efficiency of 9.58 mg of human IgG to 2 ml ofThermo Scientific AminoLink Resin using the standard coupling protocol.pHCoupling Efficiency of 9.58 mg Human IgG4 91.8%5 92.7%6 89.1%7 87.3%8* 85.3%9* 94.9%10* 98.4%* Schiff-base formation occurs readily at high pH, but reduction to stable secondaryamine bond requires subsequent incubation with sodium cyanoborohydride (AminoLinkReductant) at pH 7.2.ReferencesCheadle, C., et al. (1994). J. Biol. Chem. 269(39), 24034–24039.Cofano, F., et al. (1990). J. Biol. Chem. 265(7), 4064–4071.DeSilva, B.S. and Wilson, G.S. (1995). J. Immunol. Method 188, 9–19.Rivero-Lezcano, O.M., et al. (1994). J. Biol. Chem. 269(26), 17363–17366.Czermak, B.J., et al. (1999). J. Immunol. 162, 2321–2325.Assad, F.F., et al. (2001). J. Cell Biol. 152, 531–543.Zuk, P.A. and Elferink, L.A. (2000). J. Biol. Chem. 275(35), 26754–26764.6.Ordering InformationProduct # Description Pkg. Size44890 AminoLink Immobilization KitIncludes: AminoLink ColumnsAminoLink Coupling Buffer (pH 7.0)Quenching BufferWash BufferSodium Cyanoborohydride SolutionColumn Accessories20384 AminoLink Immobilization Trial KitIncludes: AminoLink Column Reagents and BuffersKit5 x 2 ml240 ml60 ml240 ml0.5 mlTrial Kit1 x 2 ml20381 AminoLink Coupling Resin 10 ml20382 AminoLink Coupling Resin 50 ml44892 AminoLink Reductant(Sodium cyanoborohydride)2 x 1 gH 2 NAgaroseBeadOCHAgaroseBeadNCHNaCNBH 3AgaroseBeadHCHNHYYYBeadYYProtein primary amines on antibody reactspontaneously with aldehyde groups onresin resulting in Schiff-base bondsSodium cyanoborohydridereduces Schiff base to stablesecondary amine bondMany aldehyde groups per beadand several amines per antibodyand many antibody molecules per beadThermo Scientific AminoLink Support detailed immobilization chemistry.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.15

Thermo Scientific Products for Covalent Couplingof Affinity Ligands to Chromatography SupportsUltraLink BiosupportA durable, polyacrylamide resin, activated for efficient couplingof proteins.Thermo Scientific UltraLink Biosupport is a durable, porous resin thatis activated to enable efficient and direct covalent immobilization ofproteins and other biomolecules through their primary amines for usein affinity purification procedures.Highlights:• High coupling efficiency and capacity – immobilizes proteinswith very high efficiency and coupling capacity in 1 hour• Specific and leak-proof coupling chemistry – reacts specificallywith primary amines (–NH 2 ), resulting in amide bonds that arestable for use in many affinity purification procedures; couplingreaction has no leaving group to contaminate samples• Easy to use – no pre-swelling or secondary reagents required;simply weigh the needed amount of dry support and add theligand solution to initiate coupling reaction• Flexible coupling conditions – perform immobilization reactionin any of a variety of non-amine buffers and pH levels; couplingis most efficient in buffers containing a lyotropic salt such assodium citrate; coupling compatible with or without organic solvent• Excellent reusability – prepared affinity resin can be used withtypical binding and elution procedures for more than 100 cyclesof affinity purification without significant loss of binding capacity• Durable, high-performance resin – porous beads have a 60 µmdiameter, can withstand 100 psi (6.9 bar) and allow for linearflow-through rates of 3,000 cm/hourResinBeadNOCH 3CH 2O+H 2 NProteinResinBeadONHONHProteinPierce CDI SupportsCarbonyldiimidazole-activated resins for ligand immobilization.Highlights:• Reliable coupling chemistry – immobilization occurs through thereaction of N-nucleophiles with 1,1’-carbonyldiimidazole groupsof the resin to form a stable, uncharged N-alkylcarbamate linkages• Easy-to-use – no secondary reagents needed; just wash equilibrateresin in alkaline coupling buffer and add ligand; reaction proceedsspontaneously• Stable activation – half-life of hydrolysis is much longer thanhydroxysuccinimide ester activations, making immobilizationreactions simpler to prepare and control; simplifies filtration andwashing before adding a ligand or protein• Well-defined coupling conditions – reaction is most efficient withprimary amines at pH 9-11CDI-Activated Crosslinked 6% Beaded Agarose:• Beaded agarose – the most popular resin for routine affinitypurification methods• Highly activated – at least 50 µmol 1,1’-carbonyldiimidazole (CDI)groups per milliliter of resin• Convenient form – supplied as stabilized, 50% slurry in acetoneCDI-Activated Trisacryl ® GF-2000:• Trisacryl resin – rigid, polyacrylamide matrix allows for high flow rates• Hydrophilic matrix without charge effects – provides for lownonspecific binding• Highly activated – at least 50 µmol 1,1’ -carbonyldiimidazole (CDI)groups per milliliter of resin• Convenient form – supplied as stabilized, 50% slurry in acetoneTherm ScientificUtraLink Biosupport(Azlactone Ring on Bead)AmineLigandAmide Bond Formationwith Ring OpeningResinBeadO CNON+H 2 NProteinResinBeadO CHNOProteinThermo Scientific UltraLink Biosupport immobilization chemistry.ImidazoleCarbamateAmineLigandCarbamateLinkageReferences1. Ju, T., et al. (2002). J. Biol. Chem. 277, 169–177.2. Ju, T., et al. (2002). J. Biol. Chem. 277, 178–186.3. Kornfeld, R., et al. (1998). J. Biol. Chem. 273, 23202–23210.4. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171–38176.Ordering InformationCDI immobilization chemistry.References1. Shenoy, S.K., et al. (2001). Science 294, 1307–1313.2. Richardson, R.T., et al. (2000). J. Biol. Chem. 275, 30378–30386.3. Tanaka, M, et al. (2005). PLoS Biology 3, 764–776.Product # Description Pkg. Size53110 UltraLink Biosupport (8–10 ml) 1.25 g53111 UltraLink Biosupport (50 ml) 6.25 g28388 BupH Citrate-Carbonate Buffer Packs 10 packs28386 BupH Citrate-MOPS Buffer Packs 10 packsOrdering InformationProduct # Description Pkg. Size20259 Pierce CDI (6X) Support1,1’-Carbonyldiimidazole activated crosslinked 6%beaded agaroseSupplied: stabilized in acetone slurry Agarosehydrated particle size: 45–165 µmActivation level: > 50 µmol/ml of resin10 ml20377 Pierce CDI Trisacryl Support 50 ml16For more information, or to download product instructions, visit www.thermo.com/pierce

SulfoLink Immobilization Kits and Coupling ResinCovalent immobilization of sulfhydryl-containing peptides orproteins for affinity purification.10075+TCEP+TCEPThermo Scientific SulfoLink Coupling Resin is porous, crosslinked6% beaded agarose that has been activated with iodoacetylgroups. When incubated with a solution of peptide or protein thatcontains reduced cysteine residues, the iodoacetyl groups reactspecifically and efficiently with the exposed sulfhydryls (–SH) toform covalent and irreversible thioether bonds that permanentlyattach the peptide or protein to the resin. The result is a custommadeaffinity resin for purification of antibodies, antigens and othermolecules of interest.PercentImmobilized50250-TCEPPeptide A-TCEPCalcitonin PeptideHighlights:• Specific conjugation through sulfhydryl (–SH) groups – theiodoacetyl groups react specifically with sulhydryls to formirreversible thioether bonds• Separate kits optimized for peptides or proteins – kits includeoptimized reagents for preparing peptide or protein samples forefficient immobilization• Fast – spin columns increase protocol speed; prepare andcouple samples in 2 hours (peptides) to 3.5 hours (proteins)• Flexible coupling conditions – use pH 7.5–9.0 aqueous buffers,organic solvent (e.g., 20% DMSO) or denaturant (guanidine•HCl),as needed for protein or peptide solubility during coupling reaction• Easy-to-follow instructions – streamlined protocols for samplepreparation, immobilization, and affinity purification• High capacity – immobilize 1–2 mg peptide or 2–20 mg proteinper 2–ml column of SulfoLink Coupling ResinAgaroseBeadAgaroseBead12-atomSpacerHNHNOIodoacetylGroupThermo ScientificSulfoLink Coupling ResinOISThioetherBond+PeptideHSPeptideReduced SulfhydrylMolecule+ HIImproved retention of peptides on Thermo Scientific SulfoLink Resin with TCEP.TCEP effectively reduces peptides to maximize immobilization efficiency.Two peptides (Peptide A and human calcitonin peptide) were incubated with25 mM TCEP for 30 minutes and immobilized onto SulfoLink Resin via their reducedsulfhydryl groups. Peptide A’s cysteine had oxidized during long-term storageand the calcitonin peptide contained an internal disulfide bond. Each peptide alsocontained an amine-terminal fluorescent probe by which the binding of the peptidecould be monitored during the immobilization and wash steps.ReferencesGrunwald, R. and Meissner, G. (1995). J. Biol. Chem. 270(19), 11338–11347.Seubert, P., et al. (1993). Nature 361, 260–263.Sukegawa, J., et al. (1995). J. Biol. Chem. 270(26), 15702–15706.Wisniewski, J.R., et al. (1994). J. Biol. Chem. 269(46), 29261–29264.Sakaguchi, K., et al. (2000). J. Biol. Chem. 275, 9278–9283.Tan, M., et al. (2000). Proc. Natl. Acad. Sci. USA 97, 109–114.Quill, T.A., et al. (2001). Proc. Natl. Acad. Sci. USA 98, 12527–12531.Tokumaru, H., et al. (2001). Cell 104, 421–432.Assad, F.F., et al. (2001). J. Cell Biol. 152, 531–543.Ordering InformationProduct # Description Pkg. Size44995 SulfoLink Immobilization Kit for ProteinsIncludes: SulfoLink ColumnsSulfoLink Preparation BufferSulfoLink Coupling BufferWash SolutionPhosphate Buffered Saline2-MercaptoethylamineL-CysteineSpin Desalting Columns44999 SulfoLink Immobilization Kit for PeptidesIncludes: SulfoLink ColumnsSulfoLink Coupling BufferWash SolutionPhosphate Buffered SalineBond-Breaker ® TCEPL-Cysteine20325 SulfoLink Trial KitIncludes: Pre-packed Column of SulfoLink ResinBuffers and Reagents for one proteinor peptide immobilizationKit5 x 2 ml7.5 ml500 ml120 ml1 pack5 x 6 mg100 mg5 x 5 mlKit5 x 2 ml120 ml120 ml1 pack0.5 ml100 mlTrial Kit1 x 2 ml20401 SulfoLink Coupling Resin 10 ml20402 SulfoLink Coupling Resin 50 mlImmobilized PeptideThermo Scientific SulfoLink Support immobilization chemistry.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.17

Thermo Scientific Products for Covalent Couplingof Affinity Ligands to Chromatography SupportsUltraLink Iodoacetyl ResinPolyacrylamide resin for coupling sulfhydryl-containing ligands.Thermo Scientific UltraLink Iodoacetyl Resin is a durable,porous resin that is activated to enable efficient and directcovalent immoblization of peptides and other ligands through theirsulfhydryl groups (–SH) for use in affinity purification procedures.The beaded resin is a hydrophilic copolymer of polyacrylamideand azlactone having a rigid polymeric structure with high surfacearea and pore volume. Each azlactone group has been modified toform a 15-atom spacer arm that terminates in an iodoacetyl group,which is capable of reacting with sulfhydryl groups (e.g., sidechain of reduced cysteine residues) to covalently immobilizepeptide or other sulfhydryl-containing ligands. The bead structureand efficiently coupling chemistry of Iodoacetyl-ActivatedUltraLink Support results in high protein binding capacity, highlinear flow rates, low nonspecific binding and overall superiorperformance in affinity chromatography. UltraLink Resins are idealfor medium pressure applications such as FPLC.Highlights:• High coupling efficiency and capacity – immobilizes sulfhydrylcontainingproteins or other ligands with high efficiency andcoupling capacity in 1 hour• Specific and leak-proof coupling chemistry – reacts specificallywith sulfhydryl groups (reduced thiols), resulting in thioetherbonds that are stable for use in many affinity purification procedures• Simple coupling conditions – perform immobilization reaction inany of a variety of buffers; coupling is most efficient and specificat pH 8.0–8.5; coupling compatible with or without organic solvent• Excellent reusability – prepared affinity resin can be used withtypical binding and elution procedures for many cycles of affnitypurification without significant loss of binding capacity• Durable, high-performance resin – porous beads have a 60 µmdiameter, can withstand 100 psi (6.9 bar) and allow for linearflow-through rates of 3,000 cm/hourReferences1. Ju, T., et al. (2002). J. Biol. Chem. 277, 169–177.2. Hill, K., et al. (2000). J. Biol. Chem. 275(6), 3741–3744.3. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171–38176.4. Bicknell, A.B., et al. (2001). Cell 105, 903–912.Ordering InformationProduct # Description Pkg. Size53155 UltraLink Iodoacetyl ResinSupport: UltraLink Biosupport10 mlUltraLink Iodoacetyl Micro Peptide Coupling KitEasily prepare a small-scale affinity column with sulfhydrylcontainingpeptides.The Micro Peptide Coupling Kit is a microcentrifuge spincolumn kit for immobilizing small amounts (25–250 µg) of sulfhydryl-containingpeptides (e.g., cysteine-terminated peptides) ontoa beaded porous resin to create a small, reusable affinity column.The coupling and affinity purification procedures are optimized forsmall sample volumes (200–300 µl). Wash and elution steps areachieved rapidly and efficiently with the convenient microcentrifugespin columns. Each kit contains sufficient reagents for 10 couplingreactions and 20 affinity purifications. The kit is ideal for immobilizingpeptide antigens that containing a terminal cysteine residue foruse in purifying specific antibodies from small serum, ascites orculture supernatant samples.Highlights:• Optimized for small scale – ideal for coupling small amounts(25–250 µg) of sulfhydryl-containing peptide or protein forpurification of specific antibodies from crude serum• High-performance affinity resin – uses durable, polyacrylamidebasedUltraLink Iodoacetyl Resin for specific reaction tosulfhydryl groups• Efficient coupling chemistry – immobilization efficiency > 85%(as measured with 1 hour reaction using insulin, calcitonin andosteocalcin peptides)• Fast and easy to use – perform wash and elution steps usinga microcentrifuge• Reusable – use prepared peptide resin several times with nosignificant loss of capacityOrdering InformationProduct # Description Pkg. Size20485 UltraLink Iodoacetyl Micro Peptide Coupling Kit KitSufficient materials to couple 10 sulfhydryl containingpeptides or protein and perform 20 affinity purifications.Includes: UltraLink Iodoacetyl Resin Spin Columns 10 eachEach column contains 400 ml of 25% slurryCoupling Buffer100 mlL-Cysteine•HCI100 mgWash Solution25 mlPhosphate Buffered Saline1 packIgG Elution Buffer50 mlMicrocentrifuge Collection Tubes200 eachThermo ScientificUltraLink BeadHNOI+HSPeptideThermo ScientificUltraLink BeadHNOSPeptide+ HI15-atomSpacerIodoacetylGroupThioetherBondIodoacetyl-ActivatedThermo Scientific UltraLink ResinReduced SulfhydrylMoleculeImmobilized PeptideThermo Scientific UtraLink Iodoacetyl immobilization chemistry.18For more information, or to download product instructions, visit www.thermo.com/pierce

Disulfide Reducing AgentsReduce disulfide bonds to produce sulfhydryl groups forimmobilization on Thermo Scientific SulfoLink or UltraLink Resins.Free sulfhydryls are required for immobilization onto sulfhydrylreactiveaffinity supports. Cysteines in proteins and peptidesusually exist as cystines (disulfide bridges) and must be reducedto expose sulfhydryls for coupling. Reduction can be accomplishedwith free or immobilized reducing agents. Free reducing agentsare efficient in reducing all disulfides in proteins, including thoseburied in the tertiary structure, but they must be removed from thereduced sample with a desalting column before coupling to thesupport. Immobilized reducing agents enable reduction of disulfidesand simple removal of the reduced sample from the reducingagent. This is especially helpful when reducing peptides whosesmall size prevents them from being effectively desalted.HOOPOTCEP•HClMW 286.65OHH + Cl –OHOrdering InformationOHS+ NH3 Cl –2-Mercaptoethylamine•HCIMW 113.61HSOHOHDTTMW 154.25Product # Description Pkg. Size20408 2-Mercaptoethylamine•HCl 6 x 6 mg20290 DTT, Cleland’s Reagent(Dithiothreitol)20291 Dithiothreitol (DTT) in No-Weigh Format7.7 mg DTT/tube. Makes 100 µl of 0.5 M DTT.20490 TCEP•HCl(Tris[2-carboxyethyl]phosphine hydrochloride)20491 TCEP•HCl 10 g77720 Bond-Breaker TCEP Solution, Neutral pH 5 ml77712 Immobilized TCEP Disulfide Reducing Gel 5 ml5 gSH48 tubes1 gEllman’s Reagent and Sulfhydryl Addition KitMeasure and add free sulfhydryls to ensure success ofcysteine-targeted immobilization.Ellman’s Reagent, also called DTNB, is a versatile water-solublecompound for quantifying free sulfhydryl groups in solution. Asolution of this compound produces a measurable yellow-coloredproduct when it reacts with sulfhydryls (–SH groups). By testingan unknown sample, such as a peptide having a terminal cysteineresidue, compared to a standard curve made with known amountsof free, reduced cysteine (Product # 44889), availability of reducedsulfhydryls in the sample can be determined.The Sulfhydryl Addition Kit provides the essential reagents andprocedure for creating new sulfhydryl groups on a protein orother molecule that contains available primary amines (–NH 2 ).The kit uses SATA reagent, which forms covalent bonds to primaryamines. The result is addition of a stable (capped) sulfhydrylgroup, which can later be exposed by gentle treatment withhydroxylamine, making the molecule ready for conjugation toSulfoLink Coupling Resin, UltraLink Iodoacetyl Resin or other sulfhydryl-reactiveimmobilization method.Ordering InformationProduct # Description Pkg. Size23460 Sulfhydryl Addition KitAdds free sulfhydryl groups to proteins.Includes: SATAHydroxylamine•HCl10X Conjugation Buffer StockPhosphate Buffered Saline PackDimethylformamide (DMF)Dextran Desalting ColumnColumn ExtenderEllman’s Reagent (DTNB)Cysteine•HCl H 2 O22582 Ellman’s Reagent(5,5’-Dithio-bis-[2-nitrobenzoic acid])Kit2 mg5 mg20 ml1 pack1 ml1 x 5 ml12 mg20 mg44889 Cysteine•HCl 5 g5 gTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.19

Thermo Scientific Products for Covalent Couplingof Affinity Ligands to Chromatography SupportsCarboLink Immobilization Kit and Coupling ResinsImmobilize polyclonal antibodies and other glycoproteins throughcarbohydrate groups.Thermo Scientific CarboLinkCoupling Resin and Kitsprovide for covalent immobilizationof glycoproteins andother carbohydrate-containingmolecules to beaded agarose(or polyacrylamide UltraLinkSupport) for use in affinitypurification procedures.Carbohydrate moieties inglycoproteins contain commonsugars whose cis-diol groups are easily oxidized with sodiummeta-periodate (included in the CarboLink Kit) to yield aldehydes.When incubated with the CarboLink Resin, these aldehyde groupsreact spontaneously with the hydrazide group of the activatedresin to form stable, covalent bonds. The immobilization strategy isespecially useful for glycoproteins, such as polyclonal antibodies,because it allows attachment of the molecule at domains that willnot interfere with binding sites that are critical for the intendedaffinity purification. Once a molecule is coupled, the preparedaffinity resin can be used multiple times in typical protein affinitypurification procedures.Highlights:• CarboLink Coupling Resin – hydrazide-activated crosslinked6% beaded agarose (or hydrazide-activated UltraLink Support,a beaded, polyacrylamide resin)• Efficient immobilization – couple 1–5 mg of oxidized polyclonalantibody or other glycoprotein per milliliter of resin (CarboLinkResin contains greater than 14 µmol hydrazide groups per milliliter)• Stable linkage – resonance structure of the hydrazone bondsare sufficiently stable to allow multiple rounds of affinitypurification with one batch of prepared resin; no stabilizingreductant required• Flexible and gentle coupling conditions – immobilization reactioncompleted in simple buffers (PBS or other non-amine buffer withor without organic solvent) at near-neutral pH• Ideal for polyclonal antibodies – immobilizes IgG throughcarbohydrates in the Fc region, so both antigen binding sites arefree to interact with the antigen in the mobile phase• Effective for any molecule with oxidizable sugars – first step isoxidation of the sugar groups, which allows the cis-diols of theIgG to be transformed into reactive aldehyde moieties; thesealdehydes then combine with hydrazide groups on the matrix toform stable, leak-resistant linkages• Convenient kits and product sizes – choose one- or five-columnkit containing complete sets of buffers, oxidizing reagent andversatile spin/drip columns containing the beaded agarose resin;or choose the polyacrylamide-based UltraLink Support. Bulkquantities are available for manufacturing applicationsAgaroseBead23-atomSpacerAgaroseBeadOThermo ScientificCarboLink ResinNHNH 2HydrazideGroupONHNImmobilized GlycoproteinAntibody with CarbohydrateGroups Oxidized to FormAldehyde GroupsOxidized GlycoproteinHydrazoneBondThermo Scientific CarboLink Support immobilization chemistry.References1. Kumar, P.G., et al. (2001). J. Biol. Chem. 276, 41357–41364.2. Strakova, Z., et al. (1997). Mol. Pharmacol. 51, 217–224.3. Brown, M.A., et al. (2000). J. Biol. Chem. 275, 19795–19802.4. Butko, P., et al. (1999). J. Immunol. 163, 2761–2768.5. Sequra, M., et al. (1999). Infect. Immun. 67(9), 4646Ordering InformationOHProduct # Description Pkg. Size44910 CarboLink Immobilization KitIncludes: CarboLink ColumnsCarboLink Coupling BufferCarboLink Wash BufferCarboLink OxidantSpin Desalting Columns20355 CarboLink Trial KitSufficient reagents and buffers forpreparing 1 x 2 ml immunoaffinity column.Kit5 x 2 ml250 ml100 ml5 x 5 mg5 x 5 mlTrial Kit20391 CarboLink Coupling Resin 10 ml53149 UltraLink HydrazideSupport: UltraLink BiosupportSpacer Arm: 22 atomCapacity: ≥ 15 µmol functionality/ml of resin10 ml20504 Sodium meta-Periodate 25 g20For more information, or to download product instructions, visit www.thermo.com/pierce

CarboxyLink Immobilization Kits andCoupling ResinsImmobilize peptides via carboxyl groups to create an affinity column.Thermo Scientific CarboxyLink Coupling Resin and Kits provide forcovalent immobilization of peptides or other carboxyl-containing(–COOH) molecules to a porous, beaded resin for use in affinitypurification procedures. CarboxyLink Resin is crosslinked beadedagarose (or polyacrylamide UltraLink Support) that has beenactivated with diaminodipropylamine (DADPA) to contain longspacer arms, each with a primary amine at the end. When incubatedwith the resin and the carbodiimide crosslinker EDC (included inthe CarboxyLink Immobilization Kit), carboxyl-containing moleculesbecome permanently attached to the support by stable amidebonds. Once a molecule is coupled, the prepared affinity columncan be used multiple times in typical protein affinity purificationprocedures. CarboxyLink Coupling Resins can also be used toimmobilize other kinds of molecules using alternative amine-reactivecrosslinking chemistries.Highlights:• CarboxyLink Coupling Resin – DADPA-activated crosslinked4% beaded agarose (or DADPA-activated UltraLink Support,a beaded polyacrylamide resin)• Efficient immobilization – couple 1–2 mg of peptide per milliliterof resin (CarboxyLink Agarose Resin activated with greater than16 µmol amine milliliter of resin; DADPA on UltraLink Supportactivated with greater than 40 µmol amine milliliter of resin)• Stable linkage – immoblization results in covalent attachment ofcarboxyl groups by amide bonds, allowing for multiple rounds ofaffinity purification with one batch of prepared resin• Flexible and gentle coupling conditions – immobilization reactioncompleted in simple MES or other non-amine and non-carboxyl,near-neutral buffer, with or without organic solvent.• Ideal for unmodified peptides – immobilizes peptides with highcapacity and various orientations without steric hindrance,allowing for effective use in affinity purification of specificantibodies• Convenient kits and product sizes – choose five-column kits withcomplete sets of buffers, crosslinker and versatile spin/dripcolumns containing either type of resin (agarose orpolyacrylamide) or choose stand-alone resin for other usesAgaroseBeadHNHN NH 2Immobilized DADPA(Thermo Scientific CarboxyLink Resin)AgaroseBeadHNEDC CrosslinkerHN+HNHOCovalently Immobilized Ligand(attached by amide bond and long spacer arm)OOCarboxyl Ligand(e.g., peptide C-terminus)PeptidePeptideThermo Scientific CarboxyLink Support immobilization chemistry.ReferenceYoo, B.C., et al. (2002). J. Biol. Chem. 277, 15325–15332.Ordering InformationProduct # Description Pkg. Size44899 CarboxyLink Immobilization KitIncludes: DADPA ColumnsEDCCoupling BufferWash BufferAccessories20266 CarboxyLink Coupling ResinSupport: Crosslinked 4% beaded agaroseLoading: 16–20 µmol available amino groups/ml of resin53154 Carboxylink Immobilization Kitwith UltraLink ResinIncludes: UltraLink DADPA ColumnsEDCCoupling BufferWash BufferAccessories22980 EDC1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochlorideKit5 x 2 ml5 x 60 mg500 ml120 ml25 mlKit5 x 2 ml5 x 60 mg500 ml120 ml28390 BupH MES Buffered Saline Packs 10 pack5 gTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.21

Thermo Scientific Products for Covalent Couplingof Affinity Ligands to Chromatography SupportsPharmaLink Immobilization KitImmobilize drug and metabolite ligands that contain no easilyreactive functional groups.The Thermo Scientific PharmaLink Immobilization Kit uses theMannich reaction to conjugate active hydrogen chemical groupsof ligands to beaded agarose resin for use in affinity purificationprocedures. Many small metabolites and drug molecules do notcontain available primary amines, carboxyl, sulfhydryl or otherfunctional groups that can be easily targeted for chemical conjugationto a chromatography support. However, if the compoundcontains phenolic or other moieties having active hydrogens, itcan be conjugated to PharmaLink Coupling Resin by condensationwith primary amines of the support using the formaldehyde-containingPharmaLink Coupling Reagent. Once a molecule is coupled,the prepared affinity column can be used multiple times in typicalaffinity purification procedures, such as to purify ligand-specificantibodies from sera of immunized animals.PharmaLink Coupling Resin is crosslinked beaded agarose that hasbeen activated with diaminodipropylamine (DADPA) to contain longspacer arms, each with a primary amine at the end. The Mannichreaction consists of the condensation of formaldehyde (or otheraldehyde) with ammonia and a compound containing an activehydrogen atom. Primary or secondary amines can be substitutedfor ammonia in the reaction. In the PharmaLink Kit, the primaryamine (–NH 2 ) at one end of the immobilized DADPA moleculesubstitutes for the ammonia reactant, and an active hydrogen inthe target molecule provides the other reactant for the Mannichreaction. The PharmaLink Coupling Reagent supplies the requiredformaldehyde condensation reagent.Highlights:• PharmaLink Coupling Resin – DADPA-activated crosslinked4% beaded agarose• High-efficiency coupling – PharmaLink (DADPA) Resin isactivated with greater than 16 µmol amine per milliliter of resin• Stable linkage – immobilization by the Mannich reaction resultsin covalent attachment of ligand, allowing for multiple rounds ofaffinity purification with prepared column• Flexible coupling conditions – coupling buffer is MES-bufferedsaline, pH 4.7, and ethanol can be used to maintain ligandsolubility during the immobilization reaction• Immobilizes molecules with active hydrogen groups – coupleligand containing ketones, esters, phenols, acetylenes,α-picolines, quinaldines and other groups• Ideal for immobilizing small metabolites and drug compounds –use for steroidal compounds, dyes and other organic moleculesthat contain no available “handles” for easy immobilization,or that have functional groups with low reactivity or that aresterically hinderedAgaroseBeadHNDADPA ResinAgaroseBeadAgaroseBeadHN NH 2HMolecule withActive HydrogensHNHNHNHNOHHHOHNHNPossible Immobilization ProductsHHHOHFormaldehydeMannich ReactionThermo Scientific PharmaLink Support immobilization chemistry.ROCOONNCCCHOOH C C H HC C HHOHNROHCHNRCThermo Scientific PharmaLink Support immobilization targets. Examples ofactive hydrogen functional groups that can participate in the Mannich reaction(PharmaLink Immobilization).Reference1. Rao, M.N., et al. (1997). J. Biol. Chem. 272, 24455–24460.Ordering InformationSCHHRH 2 0OHOCRCOHHCHC HProduct # Description Pkg. Size44930 PharmaLink Immobilization Kit*Includes: PharmaLink ColumnsPharmaLink Coupling BufferPharmaLink Coupling ReagentPharmaLink Washer BufferAccessoriesHKit5 x 2 ml50 ml4 ml240 ml77168 PharmaLink Coupling Reagent 4 ml* See patent information on inside back cover.22For more information, or to download product instructions, visit www.thermo.com/pierce

Pierce Maleic Anhydride PlatesProteins and other primary amine-containing compounds covalentlyattach to the microplate.Great for immobilization of compounds that do not normally stick toplain polystyrene plates.Highlights:• Spontaneously reacts with primary amines• Maleic anhydride retains its integrity and coupling availabilityfor months• Available as standard 96-well microplates and as 12 × 8-wellstrip plates• Each well activated with 200 µl reagent• Plates tested for specific signal:noise ratio and coefficient ofvariation (CV) to ensure consistent performance• Approximate binding capacity: 125 pmol biotin-pentylamineper wellReferenceBrett, P.J., et al. (2002). J. Biol. Chem. 277, 20468–20476.Ordering InformationProduct # Description Pkg. Size15110 Maleic Anhydride ActivatedClear Polystyrene 96-Well Plates15112 Maleic Anhydride ActivatedClear Polystyrene 96-Well Plates15100 Maleic Anhydride ActivatedClear Polystyrene 8-Well Strip Plates15102 Maleic Anhydride ActivatedClear Polystyrene 8-Well Strip Plates15108 Maleic Anhydride ActivatedWhite Polystyrene 96-Well Plates5 plates25 plates5 plates25 plates5 platesH 2NPeptideH 2NPeptideOOOO- OOHNPeptideHOOOHOOpH 8-9 pH 3-4OOMaleic AnhydrideActivated PlateImmobilized Peptide(ready for use in assay at pH > 7)Hydrolyzed Product(peptide released)Reaction scheme for coupling amine-containing molecules to MaleicAnhydride Plates.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.23

Thermo Scientific Products for Covalent Couplingof Affinity Ligands to Chromatography SupportsPierce Maleimide Activated PlatesA convenient alternative to amine-reactive chemistries forattaching sulfhydryl-containing compounds.Maleimide groups specifically and covalently conjugate sulfhydrylgroups at neutral pH, creating a stable thioether bond.Highlights:• Spontaneous immobilization of peptides containing reducedterminal cysteines or proteins with free sulfhydryls• Available in clear, white and black 96-well plates• Each well activated with 100 µl maleimide reagent and blockedwith 200 µl bovine serum albumin (BSA)• Plates tested for specific signal:noise ratio and coefficient ofvariation (CV) to ensure consistent performance• Approximate binding capacity: 100–150 pmol sulfhydryl-peptideper wellReaction scheme for coupling sulfhydryl-containing molecules tomaleimide-activated plates.ONOOMaleimide Activated PlateOrdering InformationNOHSPeptidepH 6.5-7.5ONSOPeptideImmobilized PeptideProduct # Description Pkg. Size15150 Maleimide ActivatedClear Polystyrene 8-Well Strip Plates5 plates15152 Maleimide ActivatedWhite Polystyrene 96-Well Plates15153 Maleimide ActivatedBlack Polystyrene 96-Well Plates5 plates5 plates24For more information, or to download product instructions, visit www.thermo.com/pierce

Antibody immobilization: choosing the best Thermo Scientific Support.AmnioLink PlusCoupling ResinUltraLinkBiosupportCarboLinkCoupling ResinsSulfoLinkCoupling ResinsOrientation Kits(Protein A or Protein G)See page 61Monoclonal AntibodiesAdvantages:• Good choice whenonly small amounts ofantibody are available• Couple over a broadpH range• Good coupling efficiencyDisadvantages:• Reduction of Schiff’s basewith sodium cyanoborohydridemay adversely affectmonoclonals• Some antibodies maybe coupled through antigen-bindingsiteAdvantages:• Good choice if antibodycan withstand 1.0 Msodium citrate or sulfate• High capacity• Fast, efficient coupling• Good, for large-scale orfast-flow applicationsDisadvantages:• Some antibodies may becoupled through antigenbindingsite• Some antibodies may precipitatein high-salt bufferAdvantages:• Correctly orients antibody• Antibody must be ableto withstand oxidationconditions• Good for antibodies withlow avidity for antigenDisadvantages:• Not all monoclonals havecarbohydrate accessiblefor coupling• Conditions necessary forcoupling may adverselyaffect some monoclonalsAdvantages:• Good choice forantibodies that haveextremely high avidityfor their antigen• Allows for gentle elutionconditionsDisadvantages:• Must first reduce antibodyprior to coupling• Not good for antibodieswith low affinity fortheir antigensAdvantages:• Allows for correctorientation of antibodies• Gentle coupling conditions• Either Protein A or G willbind most antibodiesDisadvantages:• If purifying antigen fromserum, antibodies maybind to Protein A or G andco-purify with antigen• Crosslinking results insome loss of antibodyactivityPolyclonal AntibodiesAdvantages:• Excellent couplingefficiency• Good antigen recoveryDisadvantages:• Some antibodies maybe coupled through antigen-bindingsiteAdvantages:• Good choice forlarge-scale or fast-flowapplications• High capacity• Fast, efficient couplingDisadvantages:• Some antibodies maybe coupled through antigen-bindingsite• Some antibodies mayprecipitate in high-saltbufferAdvantages:• Correctly orients antibody• Antibody must be ableto withstand oxidationconditions• Good for antibodies withlow avidity for antigenDisadvantages:• Conditions necessary forcoupling may adverselyaffect some antibodiesAdvantages:• Good choice for antibodiesthat have avidity fortheir antigen• Allows for gentle elutionconditionsDisadvantages:• Must first reduce antibodyprior to coupling• Not good for antibodieswith low affinity for theirantigensAdvantages:• Allows for correctorientation of antibodies• Gentle couple conditions• Either Protein A or G willbind most antibodiesDisadvantages:• If purifying antigen fromserum, antibodies maybind to Protein A or G andco-purify with antigen• Crosslinking results insome loss of antibodyactivityHigh-Activity AntibodiesAdvantages:• Immobilization of reducedantibody allows for gentlerelution conditionsLow-Activity AntibodiesAdvantages:• Correctly orients antibodyDisadvantages:• Conditions necessary forcoupling may adverselyaffect some monoclonalsAdvantages:• Allows for correctorientation of antibodies• Gentle couple conditions• Either Protein A orProtein G will bindmost antibodiesDisadvantages:• If purifying antigen fromserum, antibodies maybind to or Protein G andco-purify with antigen• Crosslinking results insome loss of antibodyactivityTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.25

Avidin:Biotin BindingBiotin-Binding ProteinsAvidin – The extraordinary affinity of avidin for biotin allowsbiotin-containing molecules in a complex mixture to be discretelybound with avidin. Avidin is a glycoprotein found in the egg whiteand tissues of birds, reptiles and amphibians. It contains four identicalsubunits having a combined mass of 67,000–68,000 daltons.Each subunit consists of 128 amino acids and binds one moleculeof biotin. The extent of glycosylation on avidin is high; carbohydrateaccounts for about 10% of the total mass of the tetramer.Avidin has a basic isoelectric point (pI = 10–10.5) and is stable overa wide range of pH and temperature. Extensive chemicalmodification has little effect on the activity of avidin, making itespecially useful for protein purification. However, because ofits carbohydrate content and basic pI, avidin has relatively highnonspecific binding properties.BiotinBiotin, also known as vitamin H, is a small molecule (MW 244.3)that is present in tiny amounts in all living cells. The valericacid side chain of the biotin molecule can be derivatized toincorporate various reactive groups that are used to attach biotinto other molecules. Once biotin is attached to a molecule, themolecule can be affinity-purified using an immobilized version ofany biotin-binding protein. Alternatively, a biotinylated moleculecan be immobilized through interaction with a biotin-binding protein,then used to affinity-purify other molecules that specificallyinteract with it. We offer biotin-labeled antibodies and a numberof other biotinylated molecules, as well as a broad selection ofbiotinylation reagents to label any protein.HOValeric Acid Side ChainOBiotinMW 244.3HSHHHOStreptavidin – Another biotin-binding protein is streptavidin, whichis isolated from Streptomyces avidinii and has a mass of 75,000daltons. In contrast to avidin, streptavidin has no carbohydrate andhas a mildly acidic pI (5.5). Thermo Scientific Pierce Streptavidinis a recombinant form having a mass of 53,000 daltons and anear-neutral pI. Streptavidin is much less soluble in water thanavidin. There are considerable differences in the composition ofavidin and streptavidin, but they are remarkably similar in otherrespects. Streptavidin is also a tetrameric protein, with eachsubunit binding one molecule of biotin with affinity similar to that ofavidin. Guanidinium chloride will dissociate avidin and streptavidininto subunits, but streptavidin is more resistant to dissociation.Streptavidin contains an RYD sequence similar to the RGDsequence that binds cell surface receptors. The RYD sequencecan cause background in some applications.Thermo Scientific NeutrAvidin Protein – We also offer a deglycosylatedversion of avidin, known as NeutrAvidin Protein, with amass of approximately 60,000 daltons. As a result of carbohydrateremoval, lectin binding is reduced to undetectable levels, yet biotin-bindingaffinity is retained because the carbohydrate isnot necessary for this activity. NeutrAvidin Protein offers theadvantages of a near-neutral pI (6.3) to minimize nonspecificadsorption, along with lysine residues that remain available forderivatization or conjugation. NeutrAvidin Protein yields thelowest nonspecific binding among the known biotin-bindingproteins due to its near-neutral pI and lack of both carbohydrateand RYD sequence.26For more information, or to download product instructions, visit www.thermo.com/pierce

Strength of Avidin-Biotin Interaction – The avidin-biotin complexis the strongest known noncovalent interaction (K a = 10 15 M -1 )between a protein and ligand. The bond formation between biotinand avidin is rapid and, once formed, is unaffected by extremesof pH, temperature, organic solvents and most denaturing agents.These features of avidin – features that are shared by streptavidinand NeutrAvidin Protein – make immobilized forms of the biotinbindingproteins particularly useful for purifying biotin-labeledproteins or other molecules. However, the strength of theinteraction and its resistance to dissociation make it difficult toelute bound proteins from an immobilized support. Harsh, denaturingconditions (8 M guanidine•HCl, pH 1.5 or boiling in SDS-sampleloading buffer) are required to efficiently dissociate avidin:biotincomplexes. Such conditions damage the support irreversibly sothat it cannot be reused, and denature the eluted proteins so thatthey do not maintain any biological activity.Because of these binding and elution properties, purificationsbased on avidin:biotin affinity are reserved primarily for smallscaleprocedures involving immediate analysis of the elutedsample by reducing SDS-PAGE or other denaturing method. On theother hand, it is possible to take advantage of the strong avidin:biotin binding properties in immunoprecipitation (IP) and pull-downprocedures because the immunoprecipitated “prey” protein canbe recovered using elution conditions that will notalso elute the biotinylated antibody or “bait” protein. In somesituations, it may be most appropriate to use a cleavablebiotinylation reagent to label the target molecule so that it maybe recovered from its bound state to immobilized avidin byspecific cleavage of the spacer arm between biotin and targetmolecule rather than by elution of biotin from avidin.Monomeric Avidin – Immobilized Monomeric Avidin wasdeveloped to allow the purification of fully functional biotinylatedproteins. Unlike other biotin-binding proteins that require harsh,denaturing conditions to elute and recover bound molecules,Monomeric Avidin binds reversibly to biotin and allows gentleelution and recovery of biotinylated molecules using a solution of2 mM biotin to compete for the biotin-binding sites. This makes itpossible to harness the avidin:biotin interaction as a purificationtool to recover functional proteins and other biological molecules.Biotin-Binding ProductsEach of the four biotin-binding proteins discussed is availablein a variety of immobilized formats. The support resin used forImmobilized Avidin, Streptavidin and NeutrAvidin Protein is acrosslinked 6%, beaded agarose. Immobilized Monomeric Avidinuses a crosslinked 4% beaded agarose. UltraLink Biosupport isa durable, polyacrylamide-based resin with a high surface area,large pore volume and low nonspecific binding. It is suitable forpressures up to 100 psi and linear flow rates up to 3,000 cm/hour.A biotin-binding protein immobilized on beaded agarose orUltraLink Biosupport can be used for affinity purification in acolumn or batch method. NeutrAvidin Protein and Streptavidin arealso available bound to polystyrene microplates along with a driedblocking buffer. These 96-well plates are offered in transparent,white or black plates to accommodate a variety of assay types.The plates come in two forms – regular and high-binding capacity.The high-binding capacity plates contain more of the immobilizedNeutrAvidin Protein or Streptavidin and are ideal for binding largeamounts of small, biotin-containing molecule (e.g., a biotinylatedpeptide). Streptavidin immobilized on MagnaBind Magnetic Beadsis an excellent tool for cell-sorting applications.A Comparison of Biotin-Binding ProteinsThe strong association between avidin and biotin can be used inthe field of affinity separations. By attaching avidin to a solidsupport, a biotinylated product can be anchored to the same solidsupport. The attachment is stable over a wide range of pH, saltconcentrations and temperatures. To dissociate biotin from avidin,8 M guanidine•HCl, pH 1.5 or boiling in SDS-PAGE sample buffermust be used.AvidinStreptavidinThermo ScientificNeutrAvidin ProteinMolecular Weight 67K 53K 60KBiotin-binding Sites 4 4 4Isoelectric Point (pl) 10 6.8–7.5 6.3Specificity Low High HighestAffinity for Biotin (K d ) 10 15 M 10 15 M 10 15 MNonspecific Binding High Low LowestTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.27

Thermo Scientific Products for Avidin:Biotin BindingImmobilized Avidin ProductsStrong biotin interaction creates a nearly irreversible bond.Immobilized avidin can be used in a variety of applications forthe affinity purification of biotinylated macromolecules. In onevariation, an antibody that has an affinity for a particular antigenis labeled with biotin. Cells containing the antigen are lysed, thenincubated with the biotinylated antibody to form a typical antigen/antibodycomplex. To isolate the antigen, the crude mixtureis passed through an immobilized avidin or streptavidin column,which will bind the complex. After appropriate washes, the antigencan be eluted from the column with a low pH elution buffer. Thebiotinylated antibody is retained by the column.Applications:• Binding biotinylated anti-transferrin for purifying transferrinfrom serum 1• Binding biotinylated peptides and elution with an SDS/urea solution 2• Hybridization of biotinylated RNA to its complementary DNAand binding to immobilized avidin, with subsequent elution of thesingle-stranded DNA 3• Purification of double-stranded DNA 4References1. Wilchek, M. and Bayer, E.A. (1989). Protein Recognition of Immobilized Ligands.Hutchins, T.W., ed. Alan R. Liss, Inc., pp. 83–90.2. Swack, J.A., et al. (1978). Anal. Biochem. 87, 114–126.3. Manning, J., et al. (1977). Biochemistry 16, 1364–1370.4. Pellegrini, M., et al. (1977). Nucleic Acids Res. 4, 2961–2973.5. Claypool, S.M., et al. (2002). J. Biol. Chem. 277, 28038–28050.6. Sharma, K.K., et al. (2000). J. Biol. Chem. 275, 3767–3771.Ordering InformationProduct # Description20219 Avidin Agarose ResinSupport: Crosslinked 6% beaded agaroseCapacity: ≥ 20 µg biotin/ml resin20225 Avidin Agarose ResinSupport and Capacity: Same as above20362 Avidin Agarose ColumnsSupport and Capacity: Same as abovePkg. Size5 ml5 x 5 ml5 x 1 mlImmobilized Streptavidin ProductsSame high biotin-binding affinity as avidin with low nonspecificbinding.Applications:• Purification of membrane antigens in conjunction withbiotinylated monoclonal antibodies 1,2• Cell-surface labeling with biotinylation reagents, followed byprecipitation with immobilized streptavidin 3• Purification of cell-surface glycoproteins using biotinylatedConcanavalin A 4• Recovery of single-stranded DNA for dideoxy sequencing 5References1. Gretch, D.R., et al. (1987). Anal. Biochem. 163, 270–277.2. Updyke, T.V. and Nicolson, G.L. (1984). J. Immunol. Method 73, 83–95.3. Lisanti, M.P., et al. (1989). J. Cell Biol. 109, 2117–2127.4. Buckie, J.W. and Cook, G.M. (1986). Anal. Biochem. 156(2), 463–472.5. Baqui, M., et al. (2003). J. Biol. Chem. 278, 1206–1211.6. Ellerbroek, S.M., et al. (2001). J. Biol. Chem. 276, 24833–24842.7. Huh, K-H. and Wenthold, R.J. (1999). J. Biol. Chem. 274, 151–157.8. Kilic, F., et al. (2000). Proc. Natl. Acad. Sci. USA 97, 3106–3111.9. Lesa, G.M., et al. (2000). J. Biol. Chem. 275, 2831–2836.10. Liu, L.A. and Engvall, E. (1999). J. Biol. Chem. 274, 38171–38176.Ordering InformationProduct # Description20347 Streptavidin Agarose ResinSupport: Crosslinked 6% beaded agaroseCapacity: 1–3 mg biotinylated BSA/ml resin15–28 µg biotin/ml resin20349 Streptavidin Agarose ResinSupport and Capacity: Same as above20353 Streptavidin Agarose ResinSupport and Capacity: Same as above20351 Streptavidin Agarose ColumnsSupport and Capacity: Same as above53113 Streptavidin UltraLink ResinSupport: UltraLink BiosupportCapacity: ≥ 2 mg biotinylated BSA/ml resin≥ 24 µg biotin/ml resin53114 Streptavidin UltraLink ResinSupport and Capacity: Same as above53116 Streptavidin Plus UltraLink ResinSupport: UltraLink BiosupportCapacity: ≥ 4 mg biotinylated BSA/ml resin≥ 48 µg biotin/ml resin53117 Streptavidin Plus UltraLink ResinSupport and Capacity: Same as above20357 High Capacity Streptavidin Agarose ResinSupport: Crosslinked 6% beaded agaroseCapacity: > 10 mg biotinylated BSA/ml of resin20359 High Capacity Streptavidin Agarose ResinSupport and Capacity: Same as above20361 High Capacity Streptavidin Agarose ResinSupport and Capacity: Same as above21344 MagnaBind Streptavidin BeadsSupport: 1–4 µm, iron oxide particlesCapacity: 2 µg biotin/ml beadsPkg. Size2 ml5 ml10 ml5 x 1 ml2 ml5 ml2 ml5 ml2 ml5 ml10 ml5 ml28For more information, or to download product instructions, visit www.thermo.com/pierce

Immobilized NeutrAvidin ProductsLess nonspecific binding produces cleaner results and better yields.When nonspecific binding is a problem in your application,Thermo Scientific Immobilized NeutrAvidin Products are superioralternatives to avidin or streptavidin. NeutrAvidin Biotin-BindingProtein is a modified avidin derivative that combines severalkey features to provide biotin-binding with exceptionally lownonspecific binding properties.Highlights:• Carbohydrate-free – just like streptavidin, NeutrAvidin Biotin-Binding Protein has no carbohydrate, eliminating nonspecificbinding problems due to sugars• No interaction with cell surface molecules – absence of theArg-Tyr-Asp sequence (present in streptavidin), which mimicsthe universal cell surface recognition sequence present in avariety of molecules, eliminates cross-reactivity of cell surfacemolecules• Neutral pl – with a pl of 6.3, NeutrAvidin Protein has a pl that iscloser to neutrality than avidin or streptavidin, eliminatingelectrostatic interaction that contributes to nonspecific bindingApplications:• Immunoprecipitation• Purifying proteins that bind to biotinylated ligands• Capturing biotinylated cell-surface proteins 1-3• Purifying biotinylated peptides 4References1. Conti, L.R., et al. (2001). J. Biol. Chem. 276, 41270–41278.2. Daniels, G.M. and Amara, S.G. (1998). Methods Enzymol. 296, 307–318.3. Liaw, P.C.Y., et al. (2001). J. Biol. Chem. 276, 8364–8370.4. Oda, Y., et al. (2001). Nature Biotechnology 19, 379–382.5. Hiller, Y., et al. (1987). Biochem. J. 248, 67–171.6. Butler, J.E., et al. (1992). J. Immunol. Method 150, 77–90.7. Murakami, T., et al. (2000). Proc. Natl. Acad. Sci. USA 97(1), 343–348.8. Cernuda-Morollon, E., et al. (2001). J. Biol. Chem. 276, 35530–35536.9. Hiller, Y., et al. (1987). Biochem. J. 248, 67–171.10. Kim, K., et al. (2001). J. Biol. Chem. 276, 40591–40598.11 Leighton, B.H., et al. (2002). J. Biol. Chem. 277, 29847–29855.12 Lesa, G.M., et al. (2000). J. Biol. Chem. 275, 2831–2836.13. Trotti, D., et al. (2001). J. Biol. Chem. 276, 576–582.Ordering InformationProduct # Description29200 NeutrAvidin Agarose ResinSupport: Crosslinked 6% beaded agaroseCapacity: > 20 µg or 80 nmol biotin/ml resin(approx. 1–2 mg biotinylated BSA/ml resin)29201 NeutrAvidin Agarose ResinSupport and Capacity: Same as above53150 NeutrAvidin UltraLink ResinSupport: UltraLink BiosupportCapacity: 12–20 µg biotin/ml gel53151 NeutrAvidin Plus UltraLink ResinSupport: UltraLink BiosupportCapacity: ≥ 30 µg biotin/ml gel29202 High Capacity NeutrAvidin Agarose ResinSupport: Crosslinked 6% beaded agaroseCapacity: > 75 µg biotin/ml resin> 8 mg biotinylated BSA/ml resin29204 High Capacity NeutrAvidin Agarose ResinSupport and Capacity: Same as abovePkg. Size5 ml10 ml5 ml5 ml5 ml10 mlTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.29

Thermo Scientific Products for Avidin:Biotin BindingImmobilized Monomeric Avidin and KitIdeal affinity support for gentle, reversible binding of biotinylatedproteins.Immobilized Iminobiotin and BiotinIminobiotin offers mild dissociation conditions at pH 4.NHTo break the avidin-biotin interaction, 8 M guanidine•HCl at pH 1.5or boiling in SDS-PAGE sample buffer is required. These elutionmethods may result in denaturation of the biotinylated protein andcause irreversible damage to the support. In addition, avidin orstreptavidin will be irreversibly denatured and lose the ability tobind subsequent biotinylated samples.AgaroseBeadHNHNHNOHNSNHWhen avidin is coupled to a solid support as the subunit monomer,the specificity for biotin is retained, but the affinity for biotin bindingsubstantially decreases (K a ~ 10 8 M -1 ). The Monomeric AvidinAgarose Resin and Kit can be used to bind biotinylated molecules,and the bound material can be competitively eluted using 2 mMbiotin in phosphate-buffered saline (PBS). This technique providesthe gentlest elution conditions without contamination of the avidinsubunits or substantial loss of column-binding capacity.Highlights:• Purifies biotinylated products under mild elution conditions• Can be regenerated and reused at least 10 times• Exhibits little nonspecific binding (3% or less)ReferencesBernstein, E.M., et al. (1999). J. Biol. Chem. 274(2), 889–895.Sims, K.D., et al. (2000). J. Biol. Chem. 275(7), 5228–5237.Ellerbroek, S.M., et al. (2001). J. Biol. Chem. 276, 24833–24842.Glover, B.P. and McHenry, C.S. (2001). Cell 105, 925–934.Horney, M.J., et al. (2001). J. Biol. Chem. 276, 2880–2889.Oda, Y., et al. (2001). Nature Biotechnology 19, 379–382.Schwarzman, A.L., et al. (1999). Proc. Natl. Acad. Sci. USA 96, 7932–7937.Slatin, S.L., et al. (2002). Proc. Natl. Acad. Sci. USA 99, 1286–1291.Ordering InformationProduct # Description20228 Monomeric Avidin Agarose ResinSupport: Crosslinked 4% beaded agaroseCapacity: ≥ 1.2 mg biotinylated BSA/ml resin20267 Monomeric Avidin Agarose ResinSupport and Capacity: Same as above20227 Monomeric Avidin Agarose KitSupport and Capacity: Same as aboveIncludes: 1 x 2 ml Column, Binding and Elution buffers53146 Immobilized Monomeric Avidin UltraLink ResinSupport: UltraLink BiosupportCapacity: ≥ 1.2 mg biotinylated BSA/ml resin29129 Biotin 1 gPkg. Size5 ml10 mlKit5 mlImmobilized IminobiotinIminobiotin is the guanido analog of biotin. The dissociationconstant of the avidin-iminobiotin complex is pH-dependent.At pH 9.5-11.0, the avidin-iminobiotin complex will bind tightly.At pH 4, the avidin-iminobiotin complex will dissociate. Becausedenaturing agents such as 8 M guanidine•HCI or 4 M urea arenot used in the purification, an avidin conjugate has a betterchance of maintaining its activity during purification.Use immobilized D-Biotin as an “irreversible linkage” to bindstreptavidin conjugates. The biotin-streptavidin interaction canwithstand extremes in pH, salt and detergents.ReferencesGitlin, G., et al. (1987). Biochem. J. 242, 923–926.Wood, G.S. and Warnke, R. (1981). J. Histochem. Cytochem. 29, 1196–1204.Hofmann, K., et al. (1980). Proc. Natl. Acad. Sci. USA 77(8), 4666–4668.Gao, C., et al. (1997). Proc. Natl. Acad. Sci. USA 94, 11777–11782.Hofmann, K., et al. (1980). Proc. Natl. Acad. Sci. USA 77, 4666–4668.Ordering InformationProduct # Description20221 Iminobiotin Agarose ResinSupport: Crosslinked 6% beaded agaroseSpacer: DiaminodipropylamineCapacity: ≥ 1 mg of avidin/ml resin20218 Biotin Agarose ResinSupport: Pierce CDI SupportSpacer: DiaminodipropylamineCapacity: ≥ 2 mg of avidin/ml resinPkg. Size5 ml5 ml30For more information, or to download product instructions, visit www.thermo.com/pierce

Biotinylation reagent selection guide.Product # Description Chemical Reactivity Water-Soluble Spacer Arm Length Cleavable21335* Sulfo-NHS-LC Biotin Primary Amine Yes 22.4 Å No No21338 Sulfo-NHS-LC-LC-Biotin Primary Amine Yes 30.5 Å No No21217* Sulfo-NHS-Biotin Primary Amine Yes 13.5 Å No No21331* Sulfo-NHS-SS-Biotin Primary Amine Yes 24.3 Å Yes No21442 NHS-SS-PEG 4 -Biotin Primary Amine Yes 37.9 Å Yes No21362* NHS-PEG 4 -Biotin Primary Amine Yes 29 Å No No21312* NHS-PEG 12 -Biotin Primary Amine Yes 56.0 Å No No21303 TFA-PEG 3 -Biotin Primary Amine Yes 33.4 Å No No21336 NHS-LC-Biotin Primary Amine No 22.4 Å No Yes21343 NHS-LC-LC-Biotin Primary Amine No 30.5 Å No Yes20217 NHS-Biotin Primary Amine No 13.5 Å No Yes21441 NHS-SS-Biotin Primary Amine No 24.3 Å Yes Yes21325* NHS-Chromogenic-Biotin Primary Amine No 41.0 Å No No21117 NHS-Iminobiotin TFA Primary Amine No 13.5 Å No Yes21218 PFP-Biotin Primary or Secondary Amine/RNA/DNANo 9.6 Å No Yes21219 TFP-PEG 3 -Biotin Primary Amine Yes 32.6 Å No No21901* Maleimide-PEG 2 -Biotin Sulfhydryl Yes 29.1 Å No No21911 Maleimide-PEG 11 -Biotin Sulfhydryl Yes 59.1 Å No No21900 Biotin-BMCC Sulfhydryl No 32.6 Å No Yes21334 PEG-Iodoacetyl-Biotin Sulfhydryl Yes 24.7 Å No No21333 Iodoacetyl-PEG 2 -Biotin Sulfhydryl No 27.1 Å No Yes21341 Biotin-HPDP Sulfhydryl No 29.2 Å Yes Yes21346 Amine-PEG 2 -Biotin Carboxyl ‡ Yes 20.4 Å No No21347 Amine-PEG 3 -Biotin Carboxyl ‡ Yes 22.9 Å No No21345 Pentylamine-Biotin Carboxyl ‡ Yes 18.9 Å No No28020 Biocytin Hydrazide Carbohydrate/RNA/DNA Yes 19.7 Å No No21339 Biotin Hydrazide Carbohydrate No 15.7 Å No Yes21340 Biotin-LC-Hydrazide Carbohydrate No 24.7 Å No Yes21360 Biotin-PEG 4 -Hydrazide Carbohydrate Yes 31.3 Å No No29986 Psoralen-PEG 3 -Biotin DNA/RNA Protein Yes 36.9 Å No No22020 PEG 5 -Biotin Dimer Avidin Cross-linking Yes 43.4 Å No No29987 Photoactivatable Biotin DNA/RNA Protein No 30.0 Å No Yes29982 Biotin-LC-ASA DNA/RNA Protein No 29.9 Å No Yes28022 Biocytin Hydrazide Yes 20.1 Å No No33033* Sulfo-SBED Trifunctional Yes N/A Yes No33083 Mts-Atf-LC-Biotin Trifunctional Yes N/A Yes No* Other product numbers (sizes and/or kits) also available; visit www.thermo.com/pierce for a complete listing.† Membrane permeability is implied due to a molecule’s hydrophobic/hydrophilic nature.‡ When used with EDC (Product # 22980, 22981).Membrane-Permeable †To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.31

Thermo Scientific Products for Avidin:Biotin BindingNeutrAvidin Coated Polystyrene PlatesThe high affinity of avidin for biotin, without the nonspecificbinding problems.Highlights:• Easy and gentle immobilization of biotin-containing conjugates• Lowest nonspecific binding properties of all biotin-binding proteins• NeutrAvidin Biotin-Binding Protein has no carbohydrate andan isoelectric point of 6.3• No denaturing of the protein component of a conjugate uponbinding to the plate• Ideal for binding small hydrophilic molecules (e.g., peptides) thattypically exhibit poor binding directly to polystyrene• Pre-blocked with your choice of Thermo Scientific Blocker BSAor SuperBlock ® Blocking Buffer• Available in 96- and 384-well formatsCharacteristics of avidin-biotin proteins.ProteinIsoelectricPointContainsCarbohydrateNonspecificBindingAvidin 10–10.5 Yes HighStreptavidin 5.5 No LowNeutrAvidinBiotin-Binding Protein6.3 No UltralowNet Absorbance at 450 nm1.51.00.5Purified p60 c-src Activity Detection with TK Peptide 200.00 0.05 0.10 0.15Units KinaseBiotinylated tyrosine kinase peptide 2 was added to Thermo ScientificNeutrAvidin Coated Plates and incubated for 30 minutes. Wells were washed;samples containing p60c-src tyrosine kinase were added to phosphorylate thetyrosine residue on the peptide. Anti-phosphotyrosine monoclonal antibodyconjugated to horseradish peroxidase was added. Tyrosine kinase activity wasdetected by Thermo Scientific 1-Step Turbo TMB Substrate. Kinase activitywas quantitated by comparison with a standard curve generated using thephosphorylated form of the same peptide substrate.ReferenceSingh, Y., et al. (1999). Infect. Immun. 67, 1853–1859.Ordering InformationProduct # Coating Plate Type Blocking* Binding Capacity † Pkg. Size15129 NeutrAvidin Protein, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates15127 NeutrAvidin Protein, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates15400 NeutrAvidin Protein, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~ 10 pmol biotin/well 5 plates15116 NeutrAvidin Protein, 100 µl White, 96-Well SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates15401 NeutrAvidin Protein, 50 µl White, 384-Well SuperBlock BB, 100 µl ~ 10 pmol biotin/well 5 plates15117 NeutrAvidin Protein, 100 µl Black, 96-Well SuperBlock BB, 200 µl ~ 15 pmol biotin/well 5 plates15402 NeutrAvidin Protein, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~ 10 pmol biotin/well 5 plates15123 NeutrAvidin Protein, 200 µl Clear, 96-Well Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates15128 NeutrAvidin Protein, 200 µl Clear, 8-Well Strip Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates15216 NeutrAvidin Protein, 200 µl White, 96-Well Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates15217 NeutrAvidin Protein, 200 µl Black, 96-Well Blocker BSA, 300 µl > 15 pmol biotin/well 5 plates15115 Pierce Biotin Binding Plate Sample Pack, one each of Product #s 15120, 15121, 15127, 15128 4 plates* BB = Blocking Buffer† Approximate values; plates tested for specific signal:noise and C.V.All coated 96- and 384-well plates are available in bulk quantity with bulk packagingat a discounted price.We can also custom-coat plates using a certain type of plate or a specific supplier’splate or coat with a specific surface chemistry that is not included in our standardproduct offering. Please contact our Large-Volume Custom Sales Team at 800-874-3723or 815-968-0747 for more information. Outside the United States, contact your local branchoffice or distributor.32For more information, or to download product instructions, visit www.thermo.com/pierce

NeutrAvidin High Binding Capacity (HBC)Coated PlatesUnique technology for improved assay precision.We offer researchers a wide variety of avidin-biotin products,including our exclusive Thermo Scientific Pierce NeutrAvidinCoated Plates available in a high binding capacity (HBC) format.NeutrAvidin Protein is a deglycosylated form of avidin with anear-neutral pI that results in less nonspecific binding than that ofstreptavidin or avidin. Our patent-pending plate-coating technologyoffers a NeutrAvidin HBC Plate with a wider detection limit thanour regular binding capacity plates. The standard curve exhibitsgreater linearity for detecting small biotinylated molecules suchas peptides (see Figure) and oligonucleotides, resulting in greaterassay precision. Try Thermo Scientific Pierce NeutrAvidin HBCCoated Plates for binding small biotinylated ligands and see thedifference.Highlights:• Unique plate-coating technology – results in high loading ofNeutrAvidin Protein per well• Improved sensitivity – less nonspecific binding for improved signal-to-noiseratios• Broader dynamic range – extends the quantitative range sothere’s no need for dilutions• Save time – pre-blocked plates to reduce the number of assay steps• Flexible assay formats – coated plates offered in 96- and 384-wellformats and in different colorsS/N Ratio108642HBCRBCCHC00 5 1015Biotinylated Phosphopeptide (pM/well)Comparison of Thermo Scientific NeutrAvidin High Binding Capacity (HBC)Coated Plate, NeutrAvidin Regular Binding Capacity (RBC) Coated Plates andanother supplier’s Streptavidin Coated High Binding Capacity Plates (CHC).Plates were incubated with various dilutions of biotinylated, phosphorylatedpeptide. After washing, the plates were incubated with mouse anti-phosphotyrosineantibody (1:1,000) and then detected using an anti-mouse-FITCconjugate (1:666). The Y-axis is described as the signal-to-noise (S/N) ratio.Ordering InformationProduct # Coating Plate Type Blocking* Binding Capacity † Pkg. Size15507 NeutrAvidin Protein, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 60 pmol biotin/well 5 plates15508 NeutrAvidin Protein, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 60 pmol biotin/well 5 plates15511 NeutrAvidin Protein, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~ 35 pmol biotin/well 5 plates15509 NeutrAvidin Protein, 100 µl White, 96-Well SuperBlock BB, 200 µl ~ 60 pmol biotin/well 5 plates15512 NeutrAvidin Protein, 50 µl White, 384-Well SuperBlock BB, 100 µl ~ 35 pmol biotin/well 5 plates15510 NeutrAvidin Protein, 100 µl Black, 96-Well SuperBlock BB, 200 µl ~ 60 pmol biotin/well 5 plates15513 NeutrAvidin Protein, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~ 35 pmol biotin/well 5 plates* BB = Blocking Buffer† Approximate values; plates tested for specific signal:noise and C.V.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.33

Thermo Scientific Products for Avidin:Biotin BindingPierce Streptavidin Coated Polystyrene PlatesThe specific binding affinity of streptavidin for biotin – in a microplate.Highlights:• Easy and gentle immobilization of biotin-containing conjugates• Low nonspecific binding• No denaturing of the protein component of a conjugateupon binding• Ideal for binding small biotinylated hydrophilic molecules(e.g., peptides) that typically exhibit poor binding to polystyrene• Pre-blocked with your choice of Blocker BSA or SuperBlockBlocking Buffer• Available in clear, white and black plates in 12 × 8-well strip,96-well and 384-well formatsReferencesEstrada, G., et al. (1996). Mol. Cell Probes 10, 179–185.Grobler, J.A. et al. (2002). Proc. Nat. Acad. Sci., USA 99, 6661–6666.Ordering InformationProduct # Coating Plate Type Blocking* Binding Capacity † Pkg. Size15124 Streptavidin, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates15126 Streptavidin, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 x 5 plates15120 Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates15122 Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 x 5 plates15405 Streptavidin, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~ 4 pmol biotin/well 5 plates15118 Streptavidin, 100 µl White, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates15119 Streptavidin, 100 µl Black, 96-Well SuperBlock BB, 200 µl ~ 5 pmol biotin/well 5 plates15407 Streptavidin, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~ 4 pmol biotin/well 5 plates15125 Streptavidin, 200 µl Clear, 96-Well Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates15121 Streptavidin, 200 µl Clear, 8-Well Strip Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates15218 Streptavidin, 200 µl White, 96-Well Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates15219 Streptavidin, 200 µl Black, 96-Well Blocker BSA, 300 µl ~ 10 pmol biotin/well 5 plates15115 Pierce Biotin Binding Plate Sample Pack, one each of Product #s 15120, 15121, 15127, 15128 4 plates* BB = Blocking Buffer† Approximate values; plates tested for specific signal:noise and C.V.34For more information, or to download product instructions, visit www.thermo.com/pierce

Pierce Streptavidin HBC Coated PlatesTake advantage of our technology that provides a broaderdynamic range.Thermo Scientific Pierce Streptavidin High Binding Capacity(HBC) Coated Plates are designed for binding biotinylatedoligonucleotides and peptides with higher binding efficiencythan other commercially available plates. Our proprietary coatingtechnology (patent pending) has created a streptavidin-coatedplate with four- to five-times the binding capacity of other suppliers’plates. Using our Streptavidin HBC Plate can result in an assaywith a broader dynamic range and better linearity, leading toimproved assay precision (see Figure). Try our StreptavidinHBC Coated Plate and see what has been going undetected inyour research.Highlights:• Broader dynamic range – extends the quantitative range sothere’s no need for dilutions• Better sensitivity – increased binding capacity allows directdetection of small ligands not observed with regular bindingcapacity plates• Superior assay precision – standard curve demonstratesgreater linearity• Save time – pre-blocked to reduce number of assay steps• Flexible assay formats – offered in 96- and 384-well formatsand in different colorsS/N Ratio180160140120100806040200HBCCHC0 5 10 1520 25 30Fluoresceinated Oligonucleotide (pM/well)Comparison of Thermo Scientific Pierce Streptavidin High Binding Capacity(HBC) Coated Plate with another commercially available high binding capacityplate (CHC). Plates were incubated with a biotinylated oligonucleotide, washedand probed with a complementary oligonucleotide labeled with fluorescein atvarious dilutions. The Y-axis is described as the signal-to-noise (S/N) ratio.Ordering InformationProduct # Coating Plate Type Blocking* Binding Capacity † Pkg. Size15500 Streptavidin, 100 µl Clear, 96-Well SuperBlock BB, 200 µl ~ 125 pmol biotin/well 5 plates15501 Streptavidin, 100 µl Clear, 8-Well Strip SuperBlock BB, 200 µl ~ 125 pmol biotin/well 5 plates15504 Streptavidin, 50 µl Clear, 384-Well SuperBlock BB, 100 µl ~ 60 pmol biotin/well 5 plates15502 Streptavidin, 100 µl White, 96-Well SuperBlock BB, 200 µl ~ 125 pmol biotin/well 5 plates15505 Streptavidin, 50 µl White, 384-Well SuperBlock BB, 100 µl ~ 60 pmol biotin/well 5 plates15503 Streptavidin, 100 µl Black, 96-Well SuperBlock BB, 200 µl ~ 125 pmol biotin/well 5 plates15506 Streptavidin, 50 µl Black, 384-Well SuperBlock BB, 100 µl ~ 60 pmol biotin/well 5 plates* BB = Blocking Buffer† Approximate values; plates tested for specific signal:noise and C.V.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.35

Affinity Purification of AntibodiesGeneral Purification of ImmunoglobulinsBecause antibodies have predictable structure, including relativelyinvariant domains, it has been possible to identify certain proteinligands that are capable of binding generally to antibodies, regardlessof the antibody’s specificity to antigen. Protein A, Protein Gand Protein L are three bacterial proteins whose antibody-bindingproperties have been well characterized. These proteins havebeen produced recombinantly and used routinely for affinitypurification of key antibody types from a variety of species. Agenetically engineered recombinant form of Protein A and G,called Protein A/G, is also available. These antibody-bindingproteins are available immobilized to beaded agarose resin,UltraLink Biosupport and coated onto microplates (see previoussection on Solid Supports for Affinity Purification, page 6).Antibodies specific for an antigen of interest are one of themost useful and important tools that biology researchers canpossess. The production and use of specific antibodies asdetection probes and purification ligands (i.e., immunotechnology)has revolutionized bioresearch and diagnostic technologies.Animals immunized with prepared antigens will producespecific antibodies against the antigen. When purified fromserum or hybridoma cell lines that are prepared from tissue ofthe immunized animal, the antibody can be used directly (orafter labeling with enzyme or fluorescent tags) to probe thespecific antigen in Western blotting, ELISA or a variety of otherapplications. Antibodies are most commonly purified by one oftwo affinity purification methods: general immunoglobulinpurification or specific antibody purification.Proteins A, G, A/G and L bind to antibodies at sites other than theantigen-binding domain. Therefore, these proteins can be used inpurification schemes such as immunoprecipitation (see discussionof Immunoprecipitation that follows, page 54).Proteins A, G, A/G and L have unique properties, which make eachone suitable for different types of antibody targets (e.g., antibodysubclass or animal species). It is important to realize that use ofProtein A, G or L results in purification of general immunoglobulinfrom a crude sample. Depending on the sample source, antigenspecificantibody may account for only a small portion of the totalimmunoglobulin in the sample. For example, generally only 2-5%of total IgG in mouse serum is specific for the antigen used toimmunize the animal.Immobilized Protein L, Protein A, Protein Gand Protein A/GWe offer these popular antibody-binding proteins immobilized onseveral different resins, beads and plates for use in immunoaffinitypurification techniques. All four proteins (A, G, A/G and L) areavailable as purified recombinants immobilized to crosslinked 6%beaded agarose. This is the traditional format historically used forsmall-scale column purification and immunoprecipitation methods.Our agarose resins differ from those typically offered by othersuppliers in that our immobilization method is more stable andresults in less nonspecific binding. We also offer “Plus” versionsof the Protein A, G, A/G and L agarose resins, which contain twicethe amount of protein per milliliter of resin and provide for nearlytwice the antibody binding capacity.Protein A, G, A/G and L are also available immobilized to UltraLinkBiosupport, an extremely durable, polyacrylamide-based resinwith very low nonspecific binding characteristics. The UltraLinkFormat is a perfect support for working with large volume samplesin large-scale purification methods requiring fast flow and highpressure.The interaction between the various proteins and IgG is notequivalent for all species or all antibody subclasses. The tableon the following page will help you decide which affinity proteinis best for your application.36For more information, or to download product instructions, visit www.thermo.com/pierce

Characteristics of immunoglobulin-binding proteins.RecombinantProtein LNativeProtein ARecombinantProtein ARecombinantProtein GProduction Source E. coli S. aureus E. coli E. coli E. coliMolecular Weight 35,800 46,700 44,600 21,600 50,460Number of Binding Sites for IgG 4 4 4 2 6Albumin-Binding Site No No No No NoOptimal Binding pH 7.5 8.2 8.2 5 5–8.2Binds to V L K F C F C F C F CRecombinantProtein A/GProtein A Protein G Protein A/G Protein L † AdsorbentThiophilicHuman IgG s s s s mMouse IgG s s s s sRabbit IgG s s s w mGoat IgG w s s nb sRat IgG w m m s sSheep IgG w s s nb sCow IgG w s s nb sGuinea Pig IgG s w s – sHamster IgG m m m s –Pig IgG s w s s sHorse IgG w s s – sDonkey IgG m s s – –Dog IgG s w s – sCat IgG s w s – sMonkey IgG(Rhesus)s s s – sChicken IgY nb nb nb nb mHuman IgM w nb w s mHuman IgE m nb m s –Human IgD nb nb nb s –Human IgA w nb w s mHuman IgA 1 w nb w s mHuman IgA 2 w nb w s mHuman IgG 1 s s s s mProtein A Protein G Protein A/G Protein L † AdsorbentThiophilicHuman IgG 2 s s s s mHuman IgG 3 w s s s mHuman IgG 4 s s s s mHuman Fab w w w s mHuman ScFv w nb w s mMouse IgG 1 w m m s sMouse IgG 2a s s s s sMouse IgG 2b s s s s sMouse IgG 3 s s s s sRat IgG 1 w m m s sRat IgG 2a nb s s s sRat IgG 2b nb w w s sRat IgG 2c s s s s sCow IgG 1 w s s nb sCow IgG 2 s s s nb sSheep IgG 1 w s s nb sSheep IgG 2 s s s nb sGoat IgG 1 w s s nb sGoat IgG 2 s s s nb sHorse IgG(ab) w nb w – sHorse IgG(c) w nb w – sHorse IgG(T) nb s s – sMouse IgM nb nb nb s mw = weak binding, m = medium binding, s = strong binding, nb = no binding, – means information not available* Data represent a summary of binding properties reported in the literature. Inevitably some discrepancies exist among reported values as a result of differences in bindingbuffer conditions and form of the proteins used.† Binding will occur only if the appropriate kappa light chains are present. Antibodies lambda light chains will not bind, regardless of their class and subclass.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.37

Affinity Purification of AntibodiesProtein AProtein A Characteristics and IgG Binding PropertiesProtein A is a cell wall component produced by several strains ofStaphylococcus aureus. It consists of a single polypeptide chain(MW 46.7 kDa) and contains little or no carbohydrate. 1 Protein Abinds specifically to the Fc region of immunoglobulin molecules,especially IgG. It has four high-affinity (K a = 10 8 M -1 ) binding sitesthat are capable of interacting with the Fc region of IgGs of severalspecies. 2 The molecule is heat-stable and retains its native conformationeven after exposure to denaturing reagents suchas 4 M urea, 4 M thiocyanate and 6 M guanidine hydrochloride. 3In its immobilized form (e.g., covalently coupled to beadedagarose resin), Protein A has been used extensively for isolationof a wide variety of immunoglobulins from several species ofmammals. However, the interaction between Protein A and IgGis not equivalent for all animal sources and subclasses of IgG.For example, human IgG 1 , IgG 2 and IgG 4 bind strongly to Protein A,while IgG 3 does not bind. 2 In mice, IgG 2a , IgG 2b and IgG 3 bindstrongly to Protein A, but IgG 1 (the dominant subclass in serum)binds only weakly using standard buffer conditions. Most rat IgGsubclasses bind weakly or not at all to Protein A. Despite thisvariability, Protein A is very effective for routine affinity purificationof IgG from the serum of many species. It is especially suited forpurification of polyclonal antibodies from rabbits.Weak binding of Protein A to mouse IgG 1 using traditional Tris•HClor sodium phosphate buffer systems is of particular concern andis one reason to choose Protein G when purifying mouse antibodies.However, we have developed a binding buffer that allows Protein Ato bind mouse IgG 1 nearly as well as other subclasses (see subsequentdiscussion of IgG Binding and Elution Buffers, page 44).The variable binding properties of Protein A for different subclassesof IgG can be used advantageously to separate one IgG type fromanother. Antibodies that do not bind to immobilized Protein Acan be recovered by collecting the non-bound (“flow-through”)fractions during binding and wash steps in an affinity purificationprocedure. In this way, human IgG 3 and other immunoglobulinsubclasses can be isolated from those that do bind to Protein A;however, other IgGs and serum proteins, such as albumin, will alsobe present in the non-bound fraction. Certain IgM, IgD and IgAmolecules also do not bind to Protein A and can be separated fromProtein A-binding proteins in the same manner.Immobilized Protein A ProductsThermo Scientific Pierce Immobilized Protein A is offered on severaldifferent solid supports and made available in different bindingcapacity formats, package sizes and kit formats. Protein A Agarosegenerally denotes products composed of highly purified Protein Athat is covalently coupled to crosslinked 6% beaded agarose resin.Our Protein A Agarose is available with different densities ofProtein A ligand bound to the resin. The standard version hasa binding capacity of 12–19 mg of human IgG per ml of resin, whilethe plus version has a binding capacity of > 35 mg of human IgGper ml of resin. Both resins exhibit excellent elution propertieswhen used with Pierce Buffer Systems (Figure 1), which generallyenable the resin to be regenerated and used for at least 10 roundsof purification. Supplied as a 50% resin slurry in storage buffer,Immobilized Protein A Agarose is the usual choice either forsmall-scale batch method purification procedures or for packinggravity-flow columns.Our Immobilized Protein A is also available on Trisacryl ® GF-2000,rather than agarose resin. This stable affinity support can withstandthe high-throughput volumes required in large-scale purificationprocedures. In addition, because Trisacryl GF-2000 is a hydrophilicmatrix, nonspecific binding of proteins is minimized.Thermo Scientific Pierce Protein A UltraLink Resin is anotheralternative for large-scale, high-throughput applications. UltraLinkBiosupport is composed of a hydrophilic, crosslinked bisacrylamide/azlactonecopolymer. It has an average bead diameterof 60 µm, can withstand pressures exceeding 100 psi and retainsgood chromatographic properties using flow rates up to 3,000cm/hour. Our Protein A UltraLink Resin is the ideal choice formedium pressure liquid chromatographic systems.Thermo Scientific Pierce Recombinant Protein A Agarose (Product#s 20365 and 20366) uses a genetically engineered form of ProteinA that is produced recombinantly in a nonpathogenic form ofBacillus. Non-essential regions have been removed, and fiveIgG-binding sites are included, resulting in a mass of 44.6 kDa. Someresearchers believe that the recombinant form should be used ifthe antibody preparation has strict requirements for beingenterotoxin-free. Otherwise, the native form serves as a highlyefficient means for purifying antibodies. Our RecombinantProtein A Agarose is also compatible with Pierce Binding andElution Buffers.Thermo Scientific NAb Protein A Spin Columns are available inthree package/column sizes: 10 x 0.2 ml Protein A resin in a 1 mlspin column, 5 x 1 ml resin in a 5 ml spin column, and 1 x 5 ml resinin a 22 ml spin column. For the greatest convenience, chooseNAb Protein A Plus Spin Kits (Product #s 89948 and 89978), whichinclude pre-packed columns of our Protein A Plus Agarose, as wellas binding, elution and neutralization buffers. These kits containeverything needed to isolate IgG from serum, ascites or cell culturesupernatants through a fast and simple process that requiresapproximately 30 minutes to complete. The columns in the NAbKits can each be regenerated a minimum of 10 times without a significantloss of binding capacity. NAb Protein A Spin Kits are availablein two sizes: 10 x 0.2 ml spin column kit and 2 x 1 ml spin columnkit. The spin format of these columns and kits greatly reducesthe time required to process serum, ascites and cellculture supernatants and produce a purified antibody preparation.The NAb Columns and Kits are also compatible with gravity-flowand vacuum-based purification methods.The Thermo Scientific Pierce Protein A Chromatography Cartridges(Product #s 89924 and 89925) are designed for fast, consistent separationsusing a syringe, pump or chromatography system. Theyare available in 1 ml and 5 ml sizes. The column inlet and outlet aremolded with 1/16” threads, and adaptors are included for couplingdirectly to most chromatography systems. Luer-Lok Adaptors arealso provided with the columns for simple attachment to a syringe.38For more information, or to download product instructions, visit www.thermo.com/pierce

mAU3000FTEThermo Scientific Immobilized Protein APlus ProductsAbsorbance at 280 nm250020001500100050000.05.0 10.0 15.0 20.0 25.0 30.0 35.0 mlAffinity chromatographic purification of mouse IgG from mouse ascites fluidusing Thermo Scientific Pierce Protein A Agarose and the IgG Binding andElution Buffer System. From 1 ml of mouse ascites fluid, 5.5 mg of mouse IgGwas recovered.Thermo Scientific MagnaBind Protein A (Product # 21348) isavailable to perform benchtop magnetic separations quicklyand easily. MagnaBind Beads consist of a silanized surfaceover a core of superparamagnetic iron oxide. Protein A hasbeen attached to these beads to allow IgG removal, IgGpurification or magnetic immunoprecipitation.References1. Sjoquist, J., et al. (1972). Eur. J. Biochem. 29, 572–578.2. Hjelm, H., et al. (1975). Eur. J. Biochem. 57, 395–403.3. Sjoholm, I., et al. (1975). Eur. J. Biochem. 51, 55–61.Thermo Scientific Immobilized Protein A ProductsOrdering InformationProduct # Description Pkg. Size20333 Protein A AgaroseSupport: Crosslinked 6% beaded agaroseCapacity: 12–19 mg human IgG/ml resin20334 Protein A AgaroseSupport and Capacity: Same as above20356 Protein A ColumnsSupport and Capacity: Same as above44667 Protein A IgG Purification KitIncludes: Protein A ColumnsProtein A IgG Binding BufferIgG Elution BufferDesalting Columns20338 Protein A Trisacryl ResinSupport: Trisacryl GF 2000Capacity: > 15 mg human IgG/ml resin53139 Protein A UltraLink ResinSupport: UltraLink BiosupportCapacity: > 16 mg of human IgG/ml resin5 ml25 ml5 x 1 mlKit5 x 1 ml1,000 ml500 ml5 x 5 ml5 ml5 mlTwice the amount of coupled Protein A per milliliter of resin.Ordering InformationProduct # Description Pkg. Size22810 Protein A Plus AgaroseSupport: Crosslinked 6% beaded agaroseCapacity: > 35 mg human IgG/ml resin;16–17 mg mouse IgG/ml resin22811 Protein A Plus AgaroseSupport and Capacity: Same as above22812 Protein A Plus AgaroseSupport and Capacity: Same as above89924 Pierce Chromatography Cartridges, Protein ASupport and Capacity: Same as above89925 Pierce Chromatography Cartridge, Protein ASupport and Capacity: Same as above89952 NAb Protein A Plus Spin ColumnsSupport and Capacity: Same as above89956 NAb Protein A Plus Spin ColumnsSupport and Capacity: Same as above89960 NAb Protein A Plus Spin ColumnSupport and Capacity: Same as above89948 NAb Protein A Plus Spin Purification KitIncludes: Protein A Spin ColumnsPBS Binding BufferIgG Elution BufferNeutralization BufferCollection Tubes89978 NAb Protein A Plus Spin Purification KitIncludes: Protein A Spin ColumnsPBS Binding BufferIgG Elution BufferNeutralization Buffer53142 Protein A UltraLink Plus ResinSupport: UltraLink BiosupportCapacity: > 30 mg human IgG/ml resin45202 Protein A Spin Plate96–well filter plate containing Protein A Agarosefor IgG screening1 ml5 ml25 ml2 x 1 ml1 x 5 ml10 x 0.2 ml5 x 1 ml1 x 5 mlKit10 x 0.2 ml500 ml50 ml7 ml10 x 0.2 mlKit2 x 1 ml500 ml240 ml7 ml5 ml1 plateTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.39

Thermo Scientific Products for Affinity Purification of AntibodiesMagnaBind Protein A BeadsOrdering InformationProduct # Description Pkg. Size21348 MagnaBind Protein A BeadsSupport: 1–4 µm iron oxide particlesCapacity: > 0.2 mg rabbit IgG/ml beadsProtein A Coated MicroplatesOrdering Information5 mlProduct # Description Pkg. Size15130 Protein A, Clear 96-Well PlatesCoating Volume: 100 µlBlocking: SuperBlock Blocking Buffer, 200 µlCapacity: ∼ 4 pmol rabbit IgG/well15132 Protein A, Clear 8-Well Strip PlatesSpecifications: Same as above15154 Protein A, White 96-Well PlatesSpecifications: Same as above15155 Protein A, Black 96-Well PlatesSpecifications: Same as aboveRecombinant Protein A Agarose5 plates5 plates5 plates5 platesOur recombinant form of immobilized Protein A, manufacturedwith a leak-resistant linkage.References1. Bjork, I., et al. (1972). Eur. J. Biochem. 29, 579–584.2. Goding, J.W. (1978). J. Immunol. Method 20, 241–253.3. Lindmark, R., et al. (1983). J. Immunol. Method 62, 1–13.4. Surolia, A., et al. (1982). Trends Biochem. Sci. 7, 74–76.5. Kronvall, G., et al. (1970). J. Immunol. 105,1116–1123.6. Reeves, H.C., et al. (1981). Anal. Biochem. 115 194–196.7. Kilion, J.J. and Holtgrewe, E.M. (1983). Clin. Chem. 29, 1982–1984.8. Ey, P.L., et al. (1978). Immunochemistry 15, 429–436.9. Bigbee, W.L., et al. (1983). Mol. Immunol. 20, 1353–1362.Ordering InformationProduct # Description Pkg. Size20365 Recombinant Protein A AgaroseSupport: Crosslinked 6% beaded agarose resinCapacity: ≥ 12 mg human IgG/ml resin using theIgG Buffer System20366 Recombinant Protein A AgaroseSupport and Capacity: Same as above5 ml25 mlProtein GProtein G Characteristics and IgG Binding PropertiesProtein G is a bacterial cell wall protein isolated from groupG streptococci. 1 Like Protein A from Staphylococcus aureus,Protein G binds to most mammalian immunoglobulins primarilythrough their Fc regions. Protein G binds weakly to Fab fragments. 1Sequencing of DNA that encodes native Protein G indicates thatthere are two immunoglobulin binding sites, as well as albuminand cell surface binding sites. 2 In the recombinant form ofProtein G, these albumin and cell surface binding sites havebeen eliminated to reduce nonspecific binding when purifyingimmunoglobulins. With the albumin site removed, recombinantProtein G can be used to separate albumin from crude humanimmunoglobulin samples. Recombinant Protein G has a mass ofapproximately 22 kDa. However, its apparent mass by SDS-PAGEis nearly 34 kDa.Immobilized Protein G is most commonly used for the purificationof mammalian monoclonal and polyclonal antibodies that do notbind well to Protein A. It has been reported that most mammalianimmunoglobulins bind with greater affinity to Protein G thanProtein A. 1 There are, however, species to which Protein A hasgreater affinity. 3 Protein G binds with significantly greater affinityto several immunoglobulin subclasses including human IgG 3 andrat IgG 2a . Unlike Protein A, Protein G does not bind to human IgM,IgD or IgA. 1Differences in binding characteristics between Protein A andProtein G are explained by differences in the immunoglobulinbinding sites of each protein. Although the tertiary structuresof these proteins are similar, their amino acid compositionsdiffer significantly.Inconsistency in reporting of Protein G binding characteristicsoccurs in the literature. One cause for this inconsistency likelyresults from differences in the particular source and isolationmethod used for the native Protein G characterized in each study.In addition, several methods have been used to assess relativebinding affinity including radiolabeling experiments and ELISAtechniques, the results of which are not directly comparable.Finally, significant binding differences result from differentbinding buffers used with Protein G. Optimal binding for mostimmunoglobulins to Protein G occurs in sodium acetate buffer,pH 5.0, 4 although many studies have used more neutral Tris orphosphate buffers for binding. Approximately 44% more IgG fromrat serum bound to Protein G using acetate buffer, pH 5.0 (e.g.,Protein G IgG Binding Buffer, Product # 21011) comparedto Tris•HCl pH 7.5 buffer.Immobilized Protein G ProductsLike Immobilized Protein A already discussed, Thermo ScientificPierce Immobilized Protein G is offered in several package sizes,columns and kit formats for your convenience in gravity-flow,spin and automated purification procedures. Our ImmobilizedProtein G products incorporate the recombinant form of Protein Gimmobilized to either crosslinked 6% beaded agarose or UltraLinkBiosupport. For a more detailed description of supports, see theprevious page about Pierce Immobilized Protein A Products. Bothtypes of Immobilized Protein G use coupling chemistries that are40For more information, or to download product instructions, visit www.thermo.com/pierce

leak-resistant and provide a matrix with minimal nonspecific binding.Both supports can be regenerated and reused multiple times whenstored properly.The new Thermo Scientific Pierce Protein G ChromatographyCartridges (Product #s 89926 and 89927) are designed for fast,consistent separations using a syringe, pump or chromatographysystem. They are available in 1 ml and 5 ml sizes. Column inlet andoutlet are molded with 1/16” threads and adaptors are includedfor coupling directly to most chromatography systems. Luer-LokAdaptors are also provided with the columns for simple attachmentto a syringe.For the greatest convenience, choose Thermo Scientific NAbProtein G Spin Kits (Product #s 89949 and 89979), which includepre-packed columns of our Protein G Agarose, as well as binding,elution and neutralization buffers. These kits contain everythingneeded to isolate IgG from serum, ascites or cell culture supernatantsthrough a fast and simple process that requires approximately30 minutes to complete. The columns in the NAb Kits can eachbe regenerated a minimum of 10 times without asignificant loss of binding capacity.References1. Bjorck, L. and Kronvall, G. (1984). J. Immunol. 133, 969–974.2. Guss, B., et al. (1986). EMBO J. 5, 1567–1575.3. Eliasson, M., et al. (1988). J. Biol. Chem. 263, 4323–4327.4. Åkerström, B. and Bjorck, L. (1986). J. Biol. Chem. 261, 10240–10247.Immobilized Protein G ProductsOrdering InformationProduct # Description Pkg. Size20398 Protein G AgaroseSupport: Crosslinked 6% beaded agaroseCapacity: 11–15 mg human IgG/ml resin20399 Protein G AgaroseSupport and Capacity: Same as above20397 Protein G AgaroseSupport and Capacity: Same as above89926 Pierce Chromatography Cartridges, Protein GSupport and Capacity: Same as above89927 Pierce Chromatography Cartridge, Protein GSupport and Capacity: Same as above89953 NAb Protein G Spin ColumnsSupport and Capacity: Same as above89957 NAb Protein G Spin ColumnsSupport and Capacity: Same as above89961 NAb Protein G Spin ColumnsSupport and Capacity: Same as above89949 Nab Protein G Spin Purification KitIncludes: Protein G Spin ColumnsPBS Binding BufferIgG Elution BufferNeutralization BufferCollection Tubes89979 Nab Protein G Spin Purification KitIncludes: Protein G Spin ColumnsPBS Binding BufferIgG Elution BufferNeutralization Buffer2 ml10 ml25 ml2 x 1 ml1 x 5 ml10 x 0.2 ml5 x 1 ml1 x 5 mlKit10 x 0.2 ml500 ml50 ml7 ml10 x 2 mlKit2 x 1 ml500 ml240 ml7 mlOrdering Information53125 Protein G UltraLink ResinSupport: UltraLink BiosupportCapacity: > 20 mg of human IgG/ml resin53126 Protein G UltraLink ResinSupport and Capacity: Same as above53127 Protein G UltraLink ColumnsSupport and Capacity: Same as above45204 Protein G Spin Plate96-well filter plate containing Protein G Agarosefor IgG screeningImmobilized Protein G Plus Products2 ml10 ml2 x 2 ml1 plateTwice the amount of coupled Protein G per milliliter of resin.Ordering InformationProduct # Description Pkg. Size22851 Protein G Plus AgaroseSupport: Crosslinked 6% beaded agaroseCapacity: > 20 mg human IgG/ml resin22852 Protein G Plus AgaroseSupport and Capacity: Same as above53128 Protein G Plus UltraLink ResinSupport: UltraLink BiosupportCapacity: > 25 mg human IgG/ml resinMagnaBind Protein G BeadsOrdering Information2 ml10 ml2 mlProduct # Description Pkg. Size21349 MagnaBind Protein G BeadsSupport: 1–4 µm iron oxide particlesCapacity: > 0.2 mg rabbit IgG/ml beadsProtein G Coated MicroplatesOrdering Information5 mlProduct # Description Pkg. Size15131 Protein G, Clear 96-Well PlatesCoating Volume: 100 µlBlocking: SuperBlock Blocking Buffer, 200 μlCapacity: ∼ 2 pmol rabbit IgG/well15133 Protein G, Clear 8-Well Strip PlatesSpecifications: Same as above15156 Protein G, White 96-Well PlatesSpecifications: Same as above15157 Protein G, Black 96-Well PlatesSpecifications: Same as above5 plates5 plates5 plates5 platesTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.41

Thermo Scientific Products for Affinity Purification of AntibodiesProtein A/GProtein A/G is a genetically engineered protein that combines theIgG binding profiles of both Protein A and Protein G. Protein A/Gis a gene fusion product with a mass of 50.5 kDa, designed tocontain four Fc binding domains from Protein A and two fromProtein G. Protein A/G is not as pH-dependent as Protein A(see Figure on page 45) but otherwise has the additive propertiesof Protein A and G.Protein A/G binds to all human IgG subclasses. In addition, itbinds to IgA, IgE, IgM and, to a lesser extent, IgD. Protein A/Galso binds well to all mouse IgG subclasses but does not bindmouse IgA, IgM or serum albumin. 1 This makes Protein A/G anexcellent tool for purification and detection of mouse monoclonalantibodies from IgG subclasses, without interference from IgA,IgM and murine serum albumin. Individual subclasses of mousemonoclonals are more likely to have a stronger affinity to thechimeric Protein A/G than to either Protein A or Protein G. 2Immobilized Protein A/G is an ideal choice for purification ofpolyclonal or monoclonal IgG antibodies whose subclasses havenot been determined. Overall binding capacity is greater whenpH 8.0 buffer (optimal for Protein A) is used rather than pH 5.0buffer, which is optimal for Protein G used alone. Furthermore,Thermo Scientific Pierce Protein A Binding Buffer provides forgreater binding than Tris•HCl, pH 8.0 (see description of IgGBinding and Elution Buffers on page 45).Immobilized Protein A/G ProductsLike Immobilized Protein A and G already discussed, ThermoScientific Pierce Immobilized Protein A/G is offered in severalpackage sizes, columns and kit formats for your conveniencein gravity-flow, spin and automated purification procedures.The new Thermo Scientific Pierce Protein A/G ChromatographyCartridges (Product # 89930 and 89931) are designed for fast,consistent separations using a syringe, pump or chromatographysystem. They are available in 1 ml and 5 ml sizes. Column inlet andoutlet are molded with 1/16” threads and adaptors are includedfor coupling directly to most chromatography systems. Luer-LokAdaptors are also provided with the columns for simpleattachment to a syringe.For the greatest convenience, choose Thermo ScientificNAb Protein A/G Spin Kits (Product #s 89950 and 89980) whichinclude pre-packed columns of Protein A/G Agarose, as well asbinding, elution and neutralization buffers. These kits containeverything needed to isolate IgG from serum, ascites or cellculture supernatants through a fast and simple process thatrequires approximately 30 minutes to complete. The columns inthe NAb Kits can each be regenerated a minimum of 10 timeswithout a significant loss of binding capacity.Reference1. Eliasson, M., et al. (1988). J. Biol. Chem. 263, 4323–4327Immobilized Protein A/G ProductsOrdering InformationProduct # Description Pkg. Size20421 Protein A/G AgaroseSupport: Crosslinked 6% beaded agaroseCapacity: > 7 mg human IgG/ml resin20422 Protein A/G AgaroseSupport and Capacity: Same as above89930 Pierce Chromatography Cartridges, Protein A/GSupport and Capacity: Same as above89931 Pierce Chromatography Cartridge, Protein A/GSupport and Capacity: Same as above89954 NAb Protein A/G Spin ColumnsSupport and Capacity: Same as above89958 NAb Protein A/G Spin ColumnsSupport and Capacity: Same as above89962 NAb Protein A/G Spin ColumnSupport and Capacity: Same as above89950 NAb Protein A/G Spin Purification KitIncludes: Protein A/G Spin ColumnsPBS Binding BufferIgG Elution BufferNeutralization BufferCollection Tubes89980 NAb Protein A/G Spin Purification KitIncludes: Protein A/G Spin ColumnsPBS Binding BufferIgG Elution BufferNeutralization Buffer53132 Protein A/G UltraLink ResinSupport: UltraLink BiosupportCapacity: > 20 mg human IgG/ml resin53133 Protein A/G UltraLink ResinSupport and Capacity: Same as aboveImmobilized Protein A/G Plus Products3 ml15 ml2 x 1 ml1 x 5 ml10 x 0.2 ml5 x 1 ml1 x 5 mlKit10 x 0.2 ml500 ml50 ml7 ml10 x 2 mKit2 x 1 ml500 ml240 ml7 ml2 ml10 mlTwice the amount of coupled Protein A/G per milliliter of resin.Ordering InformationProduct # Description Pkg. Size20423 Protein A/G Plus AgaroseSupport: Crosslinked 6% beaded agaroseCapacity: > 50 mg human IgG/ml resin53135 Protein A/G Plus on UltraLink SupportSupport: UltraLink BiosupportCapacity: > 28 mg human IgG/ml resinProtein A/G Coated MicroplatesOrdering Information2 ml2 mlProduct # Description Pkg. Size15138 Protein A/G, Clear 96-Well PlatesCoating Volume: 100 µlBlocking: SuperBlock Blocking Buffer, 200 µlCapacity: ∼ 5 pmol rabbit IgG/well5 plates42For more information, or to download product instructions, visit www.thermo.com/pierce

Protein LProtein L is an immunoglobulin-binding protein (35.8 kDa) thatoriginates from the bacteria Peptostreptococcus magnus, but isnow produced recombinantly. Unlike Protein A and Protein G,which bind primarily through Fc regions (i.e., heavy chain) ofimmunoglobilins, Protein L binds immunoglobulins through interactionswith their light chains. Since no part of the heavy chain is involvedin the binding interaction, Protein L binds a wider range of Ig classes thanProtein A or G. Protein L will bind to representatives of all classes of Igincluding IgG, IgM, IgA, IgE and IgD. Single-chain variable fragments(ScFv) and Fab fragments can also be bound by Protein L.Despite this wide-ranging binding capability with respect to Ig classes(which are defined by heavy chain type), Protein L is not a universalimmunoglobilin-binding protein. Binding of Protein L to immunoglobulinsis restricted to those containing kappa light chains (i.e., κ chain ofthe VL domain). 1 In humans and mice, kappa (κ) light chainspredominate. The remaining immunoglobulins have lambda (λ) lightchains. Furthermore, Protein L is effective in binding only certainsubtypes of kappa light chains. For example, it binds human VκI, VκIIIand VκIV subtypes but does not bind the VκII subtype. Binding of mouseimmunoglobulins is restricted to those having VκI light chains. 1Given these specific requirements for effective binding, immobilizedProtein L is not appropriate for general polyclonal antibody purificationfrom serum, which contains a mixture of immunoglobulins havingdifferent types of light chains. The main application for immobilizedProtein L is purification of monoclonal antibodies from ascites orculture supernatant that are known to have the VκI light chain.Protein L is extremely useful for this specific application becauseit does not bind bovine immunoglobilins, which are present in themedia serum supplement. Also, in contrast to Protein A and G,Protein L is very effective at binding IgM. Although it binds to theFab portion of the immunoglobulin monomer, Protein L does notinterfere with the antigen-binding site of the antibody. Therefore,Protein L potentially can be used in immunoprecipitation (IP)procedures.Immobilized Protein L ProductsLike Immobilized Protein A and G already discussed, ThermoScientific Pierce Immobilized Protein L is offered in severalpackage sizes, columns and kit formats for your convenience ingravity-flow, spin and automated purification procedures.The new Thermo Scientific Pierce Protein L ChromatographyCartridges (Product #s 89928 and 89929) are designed for fast,consistent separations using a syringe, pump or chromatographysystem. They are available in 1 ml and 5 ml sizes. Column inletand outlet are molded with 1/16” threads and adaptors are includedfor coupling directly to most chromatography systems. Luer-LokAdaptors are also provided with the columns for simple attachmentto a syringe.Immobilized Protein L ProductsOrdering InformationProduct # Description Pkg. Size20510 Protein L AgaroseSupport: Crosslinked 6% beaded agaroseCapacity: 5–10 mg human IgG/ml resin20512 Protein L AgaroseSupport and Capacity: Same as above89928 Pierce Chromatography Cartridges, Protein LSupport and Capacity: Same as above89929 Pierce Chromatography Cartridge, Protein LSupport and Capacity: Same as above89955 NAb Protein L Spin ColumnsSupport and Capacity: Same as above89959 NAb Protein L Spin ColumnsSupport and Capacity: Same as above89963 NAb Protein L Spin ColumnSupport and Capacity: Same as above89951 NAb Protein L Spin Purification KitIncludes: Protein L Spin ColumnsPBS Binding BufferIgG Elution BufferNeutralization BufferCollection Tubes89981 NAb Protein L Spin Purification KitIncludes: Protein L Spin ColumnsPBS Binding BufferIgG Elution BufferNeutralization BufferImmobilized Protein L Plus Products2 ml10 ml2 x 1 ml1 x 5 ml10 x 0.2 ml5 x 1 ml1 x 5 mlKit10 x 0.2 ml500 ml50 ml7 ml10 x 2 mKit2 x 1 ml500 ml240 ml7 mlTwice the amount of coupled Protein L per milliliter of resin.Ordering InformationProduct # Description Pkg. Size20520 Protein L Plus AgaroseSupport: Crosslinked 6% beaded agaroseCapacity: > 10–20 mg human IgG/ml resinProtein L Coated MicroplatesOrdering Information2 mlProduct # Description Pkg. Size15190 Protein L, Clear 96-Well PlatesCoating Volume: 100 µlBlocking: SuperBlock Blocking Buffer, 200 µl5 platesFor the greatest convenience, choose Thermo Scientific NAb Protein LSpin Kits (Product #s 89951 and 89981) which include pre-packedcolumns of Protein L Agarose, as well as binding, elution andneutralization buffers.Reference1. Nilson, B., et al. (1992). J. Biol. Chem. 267, 2234–2238.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.43

Thermo Scientific Products for Affinity Purification of AntibodiesIgG Binding and Elution Buffers forProtein A, G, A/G and LBinding and Elution Steps in Affinity PurificationAffinity purification procedures involving interaction of an antibodywith its antigen generally use binding buffers at physiologic pHand ionic strength. However, many antibody purification methodsdo not use the antibody-antigen interaction; rather, they involvebinding of antibodies by immobilized ligands that are not theantigen. In such cases, optimal binding conditions are determinedby the unique properties of the antibody-ligand interaction, whichmay be different from physiologic pH and ionic strength.Once the binding interaction occurs (i.e., the antibody is “captured”by the immobilized ligand), the support is washed with additionalbuffer to remove nonbound components of the sample. Finally,elution buffer is added to break the binding interaction and releasethe target molecule, which is then collected in its purified form.Elution buffer can dissociate binding partners by extremes of pH(low or high), high salt (ionic strength), the use of detergents orchaotropic agents that denature one or both of the molecules,removal of a binding factor, or competition with a counter ligand.In most cases, subsequent dialysis or desalting is required toexchange the purified protein from elution buffer into a moresuitable buffer for storage or use.Thermo Scientific Pierce IgG Binding and Elution Buffers have beenoptimized to provide the highest possible efficiency of IgG bindingand elution using immobilized Protein A, Protein G and Protein A/G.Use of other buffer formulations may significantly alter not only thebinding capacity but also the volumes of wash buffer required toensure good purification.General Binding and Elution Buffers for Protein A, G, A/G and LAlthough Protein A, G and A/G bind immunoglobulins adequatelyat physiologic pH and ionic strength (as with phosphate bufferedsaline, pH 7.2), optimal binding conditions are different for eachprotein. For this reason, separate IgG Binding Buffers are available foruse with each immobilized “alphabet protein” product. All our buffershave long shelf lives and are premixed for maximum ease of use.The Thermo Scientific Pierce Protein A IgG Binding Buffer is aunique, phosphate-based formulation (pH 8.0) that achieves maximumbinding capacity of IgG to immobilized Protein A. Overall IgGbinding capacity is increased with this buffer relative to traditionalbinding buffers (see Table). Most notably, the otherwise weakbinding of mouse IgG 1 is greatly improved.Thermo Scientific Pierce Protein G IgG Binding Buffer uses sodiumacetate (pH 5.0) to obtain the highest possible binding capacity ofIgG to immobilized Protein G. The binding buffer for Protein A/Gis similar to our Protein A IgG Binding Buffer. The optimal bindingwith Protein L occurs at pH 7.5; NAb Protein L Kits use phosphatebuffered saline (PBS) as the binding buffer.Generally, a Pierce Binding Buffer is used by combining it 1:1 (v/v)with clarified serum or ascites fluid. To avoid dilution, a samplecan be dialyzed into the recommended buffer. Purity of the samplesaffects the total binding capacity of Protein A, G and A/G; totalimmunoglobulin binding capacities are higher for purified and concentratedantibodies than for crude serum or dilute samples.Elution of antibodies that are bound to alphabet proteins, regardlessof the binding buffer used, is most generally accomplished using0.1 M glycine•HCl (pH 2–3) or other low pH buffer. In the vast majorityof cases, this condition breaks affinity interactions without damagingeither the immobilized protein (allowing the affinity column to bere-used) or the antibody. Our IgG Elution Buffer uses this acidic(pH 2.8) condition. With this buffer, elution of IgG is usually sharpand complete. For example, nearly all bound IgG will elute in 3 mlof buffer from a 1 ml column of Protein A.Although brief exposure of antibody to acidic elution buffer usuallyis not harmful, it is advisable to neutralize the eluate as soon aspossible after its recovery to minimize the possibility of degradation.Our IgG Elution Buffer can be neutralized easily by adding 1/10thvolume of 1 M Tris•HCl, pH 7.5–9.0. Although long-term storage ofthe purified antibody in the neutralized buffer is possible, it iscommon practice to dialyze or desalt into a buffer that is knownto be suitable for storage.Binding capacities with different buffers expressed as mg of IgG bound per 2 ml of resin.Serum Sample0.1 M Tris•HClpH 8.0Immobilized Protein A Immobilized Protein G Immobilized Protein A/GPierce Protein ABinding Buffer0.1 M Tris•HClpH 8.0Pierce Protein GBinding Buffer0.1 M Tris•HClpH 8.0Pierce Protein ABinding BufferRabbit 17.81 33.19 21.51 27.75 13.89 19.61Sheep 2.15 10.64 25.53 33.33 9.83 15.71Bovine 6.16 22.76 31.72 48.10 15.13 22.06Mouse 5.25 7.15 5.65 15.05 4.32 11.49Rat 4.99 8.30 8.43 11.80 5.20 6.66Horse 6.25 16.50 36.19 21.46 14.88 17.12Dog 35.77 22.27 13.38 20.55 21.96 24.60Chicken 0.91 1.21 1.63 7.27 1.21 4.10Pig 29.61 24.83 21.25 27.51 19.24 29.48Human 19.88 25.53 11.68 23.59 9.92 17.6744For more information, or to download product instructions, visit www.thermo.com/pierce

IgG Bound (mg)32104 5 6 7 8Buffer pHProtein AProtein GProtein A/GComparison of the binding characteristics of mouse IgG at variousbuffer pH levels.Gentle Ag/Ab Elution BufferSome antibodies are extremely labile and irreversibly denaturein the acidic conditions of the default Pierce IgG Elution Buffer.Our Gentle Ag/Ab Elution Buffer is available for such situations.This near-neutral (pH 6.55) buffer dissociates affinity-boundimmunoglobulins by ionic strength rather than by low pH. Whilebeing much less likely to degrade an antibody, it still retainsexcellent elution properties.Our researchers have tested the effect of exposure to our GentleElution Buffer on monoclonal antibody activity. In one experiment,three mouse monoclonals were incubated overnight in the GentleElution Buffer and then desalted. When analyzed in an ELISAsystem, all three monoclonals retained full antigen-bindingcapability as compared to untreated controls.9The Gentle Elution Buffer does not require neutralization and isdirectly compatible with borate, citrate and acetate buffers,including our Protein G IgG Binding Buffer. However, the GentleElution Buffer is not directly compatible with phosphate-containingbuffers, including our Protein A IgG Binding Buffer, with which itwill form an insoluble precipitate. For this reason, our GentleAg/Ab Binding Buffer, pH 8.0 is offered as a substitute for use withProtein A.Mouse IgG 1 Mild Elution BufferA unique opportunity exists in Protein A with its weaker bindingaffinity to mouse IgG 1 compared to other mouse IgG subclasses.After binding total mouse IgG to immobilized Protein A using ourProtein A IgG Binding Buffer, Thermo Scientific Pierce Mouse IgG 1Mild Elution Buffer can be used to selectively elute IgG 1 withoutaffecting the bound state of other IgG subclasses.The buffer has a mild pH (6.0–6.1) to retain better biologicalactivity in both the recovered antibody and the immobilizedProtein A. Neutralization or desalting of the collected IgG 1 is notnecessary to retain activity. This advantage is especially importantwhen isolating potentially fragile monoclonal IgG 1 antibodies. Becausethe majority of mouse monoclonals are of the IgG 1 subclass, this bufferhas many applications in the production of monoclonal antibodies.After eluting the IgG 1 , other bound IgGs can be eluted usingstandard IgG Elution Buffer. Our Protein A Binding Buffer and bothIgG and IgG 1 Mild Elution Buffers are available as a kit. The systemenables quick, clean and mild isolation of mouse IgG 1 from serum,ascites or hybridoma culture supernatant.Ordering InformationProduct # Description Highlights Pkg. Size54200 Protein A/G IgG Binding Buffer • Ensures maximum recovery of IgG from immobilized Protein A/G 240 ml2100121007210192101121004210092102021012210302102721013Protein A IgG Binding Buffer• High-yield isolation of Mouse IgG 1 using Protein A columns• Premixed and easy to useProtein G IgG Binding Buffer • Ensures maximum recovery of IgG from immobilized Protein G 1 L3.75 LIgG Elution Buffer • High-yield isolation of IgG from Immobilized Protein A and Protein G 1 L3.75 LGentle Ag/Ab Binding Buffer pH 8.0Gentle Ag/Ab Elution Buffer pH 6.6• Specially formulated and prefiltered• Eliminates use of harsh acidic elution conditions• Specially formulated for neutral elutions• Not compatible with phosphate buffers21016 IgM Binding Buffer • Specially formulated for optimal binding of mouse IgM 800 ml21017 IgM Elution Buffer • Specially formulated for optimal recovery of mouse IgM 500 ml21018 MBP Column Preparation Buffer • Specially formulated for use with Immobilized MBP and IgM Purification Kit 50 ml21034 Mouse IgG 1 Mild Elution Buffer • Separate IgG 1 from other IgG subclasses 500 ml21033 Mouse IgG 1 Mild Binding and Elution Buffer KitIncludes: Pierce Protein A IgG Binding BufferMouse IgG 1 Mild Elution BufferPierce IgG Elution Buffer• Complete kit to allow mouse IgG 1 to be separated from other mouseIgG subclasses1 L3.75 L1 L3.75 L100 ml500 ml3.75 LKit1 L500 ml1 LTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.45

Thermo Scientific Products for Affinity Purification of AntibodiesMelon Gel Purification ProductsThermo Scientific Melon Gel Products provide an exciting newapproach to purifying monoclonal and polyclonal antibodies fromserum, tissue culture supernatant and ascites fluid. Melon Gelworks with antibodies from a variety of species and subclasses,many of which do not purify efficiently with Protein A or Protein G.Because Melon Gel is not a bind-and-release support, it isextremely fast and gentle to your antibodies, resulting in antibodypreparations of high purity and high activity!H -L -Human Rabbit GoatS M A1 A2 S M A1 A2 S M A1 A2How does it work?Melon Gel contains a proprietary ligand that retains most proteinfound in serum, ascites and culture supernatants, while allowingIgG to pass through the support and be collected in the flowthroughfraction. The resulting recovery and purity of the IgGisolated by this method rivals that obtained from the same samplesusing bind-and-release supports such as Protein A or Protein G.Highlights:• Simple, one-step protocol – no tedious binding, washing, andmultiple elution steps• Rapid purification – purifies antibodies from serum four to sixtimes faster than Protein A or G methods• High recovery and purity – antibodies from many species arerecovered with greater than 90% yield and greater than 80% purity• Robust purification – works with a wide range of antibodiesincluding many that do not purify well on Protein A or Protein G• Gentle purification – No harsh elution conditions meansantibodies retain more activity• Reusable support – Melon Gel Support can be used for multipleantibody purifications• Available in various formats – spin columns, purification kits andchromatography cartridges for antibody purification from serum,ascites and culture supernatantSeveral kits and package sizes of Melon Gel Resin are available.All sizes use the same formulation of Melon Gel Resin, PurificationBuffer and Regenerant Solution but have slightly different protocolsbased on the most common application for each package size.Components of any package size can be used at an appropriatescale with any one of the procedures. Also available is a TechTip for using Melon Gel Resin to remove BSA or gelatin fromcommercially-supplied antibodies so that the antibody can be biotinylatedor otherwise labeled using amine-reactive chemistries.Thermo Scientific Melon Gel efficiently purifies IgG from human, rabbit, andgoat serum. Starting serum samples and resulting purification products wereelectrophoresed by SDS-PAGE and stained with Thermo Scientific GelCodeBlue Stain. S = serum sample, M = Melon Gel-purified product, A1 and A2 =successive elution fractions from Protein A purification. H and L denote antibodyheavy and light chain bands in the reducing gel. Similar results were obtainedwhen comparing against Protein G purification.Percentage9080706050403020100GoatHumanSpeciesRabbitMelon Gel Protein A Protein GThermo Scientifc Melon Gel provides better purity than Protein A and G.Percent purity was determined by purifying IgG from goat, human and rabbitserum using Melon Gel, Protein A and Protein G. The products were separatedby SDS-PAGE. Purity was calculated by determining the intensities of all bandsvia fluorescence scanning and densitometry.100908070Percentage6050403020100HumanMouseSpeciesRabbitMelon Gel Protein A Protein GThermo Scientific Melon Gel provides better recovery than Protein A and G.Percent recovery was determined by applying human, mouse and rabbit IgGto Melon Gel, Protein A and Protein G, and then comparing the absorbance at280 nm of the original samples against that of the purified sample.46For more information, or to download product instructions, visit www.thermo.com/pierce

IgG purification performance of the Thermo Scientific Melon Gel System,Protein A and Protein G.1 2 3 4 5 6 7 8Source Melon Gel Protein A Protein GHuman H H HMouse H H HRabbit H H HRat H L MGoat H L MCow M L HSheep M L HHorse H L HGuinea Pig H H LPig H H LChicken N N NHamster H M MDonkey H M HH = high recovery, M = medium recovery, L = low recover, N = no recoveryTransferrinAlbuminHeavy ChainLight Chain1 2 3 4 5 6Thermo Scientific Melon Gel offers better recovery and purity from cellculture supernatant than Protein G. IgG was purified from cell culturesupernatant containing 10% fetal bovine serum (FBS), resolved by SDS-PAGEand detected with Imperial Protein Stain † (Product # 24615). Lane 1. Original cellculture supernatant, Lane 2. IgG after precipitation with Saturated AmmoniumSulfate (Product # 45216), Lane 3. Melon Gel-purified IgG and Lane 4–6. ProteinG-purified IgG.TransferrinAlbuminHeavy ChainLight ChainThermo Scientific Melon Gel offers better recovery and purity fromascites fluid than Protein G. IgG 1 was purified from ascites fluid, resolved bySDS-PAGE and detected with Thermo Scientific GelCode Blue Stain Reagent(Product # 24590). Lane 1. Original ascites fluid, Lane 2. treated with AscitesConditioning Reagent (Product # 45219) and purified with Thermo ScientificMelon Gel, Lane 3. Protein G column flow-through, Lanes 4–5. Protein G columnwashes and Lane 6–8. Protein G column elutions. NOTE: The Protein G-purifiedsample is contaminated with transferrin (Lanes 6–8). The Melon Gel-purifiedsample is not (Lane 2).Ordering InformationProduct # Description Pkg. Size45206 Melon Gel IgG Spin Purification Kit †Sufficient to purify up to 25 mg of IgG from serum.Includes: Melon GelMelon Gel Purification BufferSpin ColumnsCollection Tubes45212 Melon Gel IgG Spin Purification KitSufficient to purify up to 2 g of IgG from serum.Includes: Melon Gel SupportMelon Gel Purification BufferMelon Gel Regenerant(dry mix; reconstitute to 1 L)45214 Melon Gel Monoclonal IgG Purification KitSufficient to purify IgG from up to 1 L of cell culturesupernatant or up to 200 ml of ascites fluid.Includes: Melon Gel SupportMelon Gel Purification BufferMelon Gel Regenerant45216 Saturated Ammonium Sulfate SolutionFor use with the Melon Gel Monoclonal IgGPurification Kit and Melon Gel IgG Purification Kitsfor Sera or as a general-purpose salting-out agentfor protein-preparation applications.45219 Ascites Conditioning ReagentFor use with the Melon Gel Monoclonal IgGPurification Kit.Use of this reagent is described in the instructionsprovided with Product # 45214.Kit3 ml100 ml27Kit5 ml100X, dry mixKit200 ml100X, dry mix5X, dry mix1 L5 ml89932 Pierce Chromatography Cartridges, Melon Gel 2 x 189933 Pierce Chromatography Cartridges, Melon Gel 1 x 545208 Melon Gel Spin Kit Plate96-well filter plate for IgG screening2 plates89972 Melon Gel Purification Buffer 1 pack89973 Melon Gel Regenerant 1 pack† See patent information on inside back cover.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.47

Thermo Scientific Products for Affinity Purification of AntibodiesThiophilic Gel Antibody PurificationThiophilic adsorption is a low-cost, efficient alternative toammonium sulfate precipitation for immunoglobulin purificationfrom crude samples. Ammonium sulfate precipitation must befollowed by several additional steps to completely removecontaminants in crude samples. Thiophilic adsorption is a simple,rapid, one-step method for antibody purification from serum, ascitesor tissue culture supernatant.Thiophilic adsorption is a highly selective type of lyotropicsalt-promoted protein:ligand interaction phenomenon that hasbeen studied extensively by Porath and co-workers and otherresearchers. 1 This interaction is termed thiophilic because it isdistinguished by proteins that recognize a sulfone group in closeproximity to a thioether. Thiophilic adsorption incorporates propertiesof both hydrophobic and hydrophilic adsorption. However, incontrast to strictly hydrophobic systems, thiophilic adsorption isnot strongly promoted by high concentrations of sodium chloride.Instead, thiophilic adsorption is promoted by increasedconcentrations of water-interacting, non-chaotropic salts such aspotassium and ammonium sulfate.Thermo Scientific Pierce Thiophilic Adsorbent is 6% beadedagarose modified to contain simple sulfone/ thioether groups (seestructure at right). Our Thiophilic Adsorbent has a high bindingcapacity (20 mg of immunoglobulin per ml of resin) and broadspecificity toward immunoglobulins derived from various animalspecies. Notably, thiophilic adsorption is one of few methodsavailable for purification of IgY from chicken (see also subsequentdiscussion of IgY purification). Among human serum proteins,immunoglobulins and α2-macroglobulins are preferentially boundby our Thiophilic Adsorbent. 2Purification using Pierce Thiophilic Adsorbent results in goodprotein recovery with excellent preservation of antibody activity.Sample preparation requires the addition of 0.5 M potassiumsulfate to the serum, ascites or culture fluid. Greater specificityfor immunoglobulins is obtained if the sample is buffered at pH 8.0.The gentle elution conditions (e.g., 50 mM sodium phosphate,pH 7–8) yield concentrated, essentially salt-free, highly purifiedimmunoglobulins at near neutral pH.After use, our Thiophilic Adsorbent can be regenerated by treatmentwith guanidine•HCl. Our data indicate that the Adsorbentcolumn can be used at least 10 times without significant loss ofbinding capacity.Our Thiophilic Purification Kit includes 4 x 3 ml prepackedcolumns of Pierce Thiophilic Adsorbent, binding and elutionbuffers, column storage buffer, and guanidine•HCl for use incolumn regeneration. This simple, one-step method eliminatesthe need for post-treatment of the sample before storage orsubsequent conjugation to enzymes for use in immunoassays.Suggested applications:• Efficient and selective isolation of immunoglobulins from humanserum under mild conditions 1• Convenient and fast method for purification of mousemonoclonals from the culture media of cloned cells or fromascites fluid 2• Selective removal of immunoglobulins from fetal calf serum –useful for cell culture in monoclonal antibody production 3• Rapid, straightforward procedure yielding essentially pureimmunoglobulins from crude rabbit serum 4• Purification of IgY from chicken 5• Large-scale purification for biotechnology applicationsAgaroseBeadOOSStructure of Thermo Scientific Pierce Thiophilic Adsorbent.References1. Porath, J., et al. (1985). FEBS Lett. 185, 306–310.2. Belew, M., et al. (1987). J. Immunol. Method 102, 173–182.3. Hutchens, T.W. and Porath, J. (1987). Biochemistry 26, 7199–7204.4. Lihme, A. and Heegaard, P.M.H. (1990). Anal. Biochem. 192, 64–69.5. Unpublished internal Pierce documents.OSOH48For more information, or to download product instructions, visit www.thermo.com/pierce

Pierce Thiophilic Adsorbent and Purification KitEconomical purification of mouse antibodies from ascites fluid.Highlights:• Binds to Fab and F(ab´) 2 fragments• Binds to ScFv 1• High-capacity (20 mg/ml), good protein recovery and retentionof antibody function• Broad specificity toward immunoglobulins derived from variousanimal species (see Table)• Binds chicken IgY (also called IgG)• Simple, rapid, one-step purification for monoclonal antibodiesfrom ascites; easy to scale up• Used to enrich the immunoglobulin fraction from serum or tissueculture supernatant• Efficient alternative to ammonium sulfate precipitation forenriching antibodies from crude samples• Gentle elution conditions yield concentrated, salt-freeimmunoglobulin at near neutral pH• High degree of purityReferences1. Schulze, R.A., et al. (1994). Anal. Biochem. 220, 212–214.Palmer, D.A., et al. (1994). Anal. Biochem. 222, 281–283.Porath, J., et al. (1985). FEBS Lett. 185, 306–310.Hutchens, T.W. and Porath, J. (1986). Anal. Biochem. 159, 217–226.Belew, M., et al. (1987). J. Immunol. Method 102, 173–182.Hutchens, T.W. and Porath, J. (1987). Biochemistry 26, 7199–7204.Nopper, B., et al. (1989). Anal. Biochem. 180, 66–71.Harsay, E. and Schekman, R. (2002). J. Cell Biol. 156(2), 271–85.Koustova, E. et al. (2001). J. Clin. Invest. 107(6), 737–44.Suh, J.S., et al. (1998). Blood. 91(3), 916–22.Ordering InformationProduct # Description Pkg. Size20500 Pierce Thiophilic Adsorbent 10 ml44916 Pierce Thiophilic Purification Kit †Includes: Thiophilic Adsorbent ColumnsBinding BufferElution BufferColumn Storage Buffer (2X)Guanidine•HCl CrystalsColumn Extenders† See patent information on inside back cover.Kit4 x 3 ml1,000 ml1,000 ml100 ml230 gBinding characteristics of Thermo Scientific Pierce Thiophilic Adsorbent.SpeciesTotal A 280 Boundfrom 1 ml Serum% Purity by HPLCHuman 4.8 70Mouse 8.6 63Mouse IgG 1 11.6 92Mouse IgG 2a 9.3 88Mouse IgG 2b 9.8 97Mouse IgG 3 10.7 94Rat 13.0 79Bovine 17.9 90Calf 11.1 89Chicken 5.2 76Dog 12.2 91Goat 17.3 92Guinea Pig 11.1 71Horse 13.0 93Pig 21.1 90Rabbit 6.7 84Sheep 12.3 89To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.49

Thermo Scientific Products for Affinity Purification of AntibodiesIgM PurificationStructure of IgMIgM is a high molecular mass glycoprotein (900–950 kDa) with acarbohydrate content of approximately 12%. This antibody is foundat concentrations of 0.5–2 mg/ml in serum. 1 In vivo, IgM has a halflife of five days, and its catabolism is two- to three-fold greaterthan that of IgG.In the sera of mammals, birds and reptiles, IgM has a pentamericstructure. However, mouse and human IgM structures differ in thelocation of disulfide bridges that link monomers together to formthe pentamer (Figure). 2 Disulfides are arranged in series in mouseIgM and in parallel in human IgM.Challenges to IgM PurificationProtein A binds IgM poorly, in part because binding sites on the Fcregion of the monomers are sterically hindered by the pentamericstructure of IgM. Until recently, no readily available affinitychromatography product existed for one-step IgM purification.Standard methods for IgM purification generally are multi-step,tedious processes or they are not effective for removing all of themajor impurities present in IgM samples. 3Traditionally, IgM was purified by ammonium sulfate precipitationfollowed by gel filtration, ion exchange chromatography or zoneelectrophoresis. 4 Other methods that have been used includeuse of DEAE cellulose, 5 immobilized DNA 6 and a combination ofammonium sulfate precipitation and subsequent removal of IgGwith Protein A or G. 3IgM purification with Pierce Immobilized Mannan Binding Proteinis temperature- and calcium-dependent. Binding and washingsteps are performed at 4°C in 10 mM Tris•HCl (pH 7.4) buffer containingsodium chloride and 20 mM calcium chloride. Elution ismade at room temperature in a similar Tris buffer, except that itcontains EDTA and is devoid of calcium chloride. An ImmobilizedMBP Column can be regenerated at least 10 times with no apparentloss of binding capacity.Immobilized MBP is available in both beaded agarose andUltraLink Biosupport formats. Binding, elution and column preparationbuffers are also available. The IgM Purification Kit containssufficient buffers to perform 10 purifications using a 5 ml column ofImmobilized MBP. The kit is easy to use and yields 90% puremouse IgM (from ascites) with a very simple protocol.Human IgMNethery, et al. developed an IgM affinity purification methodusing C1q, a 439 kDa complement component that recognizescarbohydrate on cell surfaces. 7 This temperature-dependentbinding method yielded relatively pure IgM. However, co-purificationof IgG was a problem, and C1q is expensive and difficultto purify.Immobilized Mannan Binding ProteinTo develop an effective affinity matrix, our scientists examinedC1q and another similarly structured protein, mannan bindingprotein (MBP). Serum MBP, like C1q, is capable of initiatingcarbohydrate-mediated complement activation. MBP is a mannoseand N-acetylglucosamine-specific lectin found in mammalian sera,and it has considerable structural homology to C1q. 8 MBP subunitsare identical, each with molecular mass of approximately 31 kDa(C1q has six each of three different polypeptide subunits of molecularmass 24–28 kDa). Studies in our labs show that MBP does notbind F(ab´) 2 and Fab.We have developed an easy-to-use Thermo Scientific PierceImmobilized Mannan Binding Protein and Buffer System to purifyIgM. It is most effective for purifying mouse IgM from ascites.Purified IgM can be obtained from a single pass over the affinitycolumn. Human IgM will bind to the support, albeit with slightlylower capacity, and yield a product at least 88% pure as assessedby HPLC. The purification of IgM from other species and mouseserum has not yet been optimized.Mouse IgMStructure of IgM, adapted from Matthew and Reichardt. 8References1. Milstein, C., et al. (1975). Biochem. J. 151, 615–624.2. Coppola, G., et al. (1989). J. Chromatogr. 476, 269–290.3. Fahey, J. and Terry, E. (1967). Handbook of Experimental Immunology,Chapter 8. D.M. Weir, Ed. Blackwell, Oxford, U.K.4. Cambier, J. and Butler F. (1974). Prep. Biochem. 4(1), 31–46.5. Abdullah, M., et al. (1985). J. Chromatogr. 347, 129–136.6. Nethery, A., et al. (1990). J. Immunol. Method 126, 57–60.7. Ohta, M., et al. (1990). J. Biol. Chem. 264, 1980–1984.8. Matthew, W. and Reichardt, L. (1982). J. Immunol. Method 50, 239–253.50For more information, or to download product instructions, visit www.thermo.com/pierce

Immobilized MBP and IgM Purification KitEasy IgM purification with guaranteed 88% pure mouse IgM!Absorbance at 280 nm(percent of max.)1008060402000 10 20 30 40 50Time (min.)Demonstration of the high purity of MBP-purified IgM from mouse ascites.The bound material from mouse ascites was eluted from the 5 ml MBP columnas described in the Standard Protocol. The highest 280 nm absorbing fractionfrom the elution was chromatographed using the conditions described in theinstructions.IgMReferencesNethery, A., et al. (1990). J. Immunol. Method 126, 57–60.Ohta, M., et al. (1990). J. Biol. Chem. 265, 1980–1984.Nevens, J.R., et al. (1992). J. Chromatogr. 597, 247–256.Ordering InformationProduct # Description Pkg. Size22212 Immobilized Mannan Binding ProteinCapacity: ~1 mg IgM/ml of resin44897 IgM Purification KitIncludes: Immobilized MBP ColumnIgM Binding BufferIgM Elution BufferMBP Column Preparation BufferColumn Extender10 mlKit5 ml800 ml500 ml50 ml21016 IgM Binding Buffer 800 ml21017 IgM Elution Buffer 500 ml21018 MBP Column Preparation Buffer 50 ml53123 UltraLink Immobilized Mannan Binding ProteinCapacity: > 0.75 mg IgM/ml of resin5 mlIgA PurificationHuman IgA PurificationJacalin is an α-D-galactose binding lectin extracted from jack-fruitseeds (Artocarpus integrifolia). The lectin is a glycoprotein ofapproximately 40 kDa composed of four identical subunits. Jacalinimmobilized on supports such as agarose has been useful forthe purification of human serum or secretory IgA 1 . IgA can beseparated from human IgG and IgM in human serum or colostrum. 1IgD is reported to bind to jacalin. 2 Immobilized jacalin is also usefulfor removing contaminating IgA from IgG samples.Binding of IgA to immobilized jacalin occurs at physiologic pHand ionic strength, as in phosphate buffered saline (PBS). Elutionof bound IgA occurs with competitor ligand (e.g., 0.1 M melibioseor 0.1 M α-D-galactose) in PBS. We offer immobilized jacalin oncrosslinked 6% agarose.References1. Roque-Barreira, M.C. and Campos-Neto, A. (1985). J. Immunol. Method 134(30), 1740–1743.2. Aucouturier, P., et al. (1987). Mol. Immunol. 24(5), 503–511.Immobilized JacalinIdeal for human IgA purification.Highlights:• Ideal for preparing human IgA that is free of contaminating IgG• Found to bind human IgA 1 , but not human IgA 2 – useful forseparating the two subclassesReferencesKumar, G.S., et al. (1982). J. Biosci. 4, 257–261.Roque-Barreira, M.C. and Campos-Neto, A. (1985). J. Immunol. 134, 1740–1743.Mestecky, J., et al. (1971). J. Immunol. 107, 605–607.Van Kamp, G.J. (1979). J. Immunol. Method 27, 301–305.Kondoh, H., et al. (1986). J. Immunol. Method 88, 171–173.Ordering InformationProduct # Description Pkg. Size20395 Immobilized JacalinCapacity: 1–3 mg human IgA/ml of resinSupport: Crosslinked 6% beaded agaroseLoading: 4.5 mg of jacalin/ml of resin5 mlTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.51

Thermo Scientific Products for Affinity Purification of AntibodiesChicken IgY PurificationProperties of IgYChickens produce a unique immunoglobulin molecule called IgY.There are several advantages to production and use of IgY overmammalian immunoglobulins. With regard to production, raisingand immunizing chickens is relatively simple, chickens are morelikely to produce an immune response to conserved mammalianprotein antigens, and chickens produce 15- to 20-times moreantibody than rabbits.Most importantly, IgY is naturally packaged at high concentrationsin egg yolks, making repeated collection of antibody fromimmunized hens noninvasive. A single egg yolk from an immunizedchicken contains approximately 300 mg of IgY. Whole eggs orseparated egg yolks can be collected and stored frozen for laterextraction of antibody.Other advantages of IgY for use in immunoassays are that it doesnot bind rheumatoid factor or other anti-mammalian IgGs, does notactivate complement, and generally has much lower probability ofnonspecific binding to mammalian tissues and extracts.IgY Purification MethodsOne challenge with regard to IgY is that it can be difficult to purify.Protein A, Protein G and other Fc-binding proteins do not bind IgY.Thermo Scientific Pierce Thiophilic Adsorbent (see page 48)enables moderate yields of fairly pure IgY from serum and otherfluids. However, complete procedures for our Thiophilic Adsorbenthave not been developed for use with egg yolks, which have very highlipid concentrations.Thermo Scientific Pierce Chicken IgY Purification Kits were specificallydeveloped for efficient purification of IgY from egg yolks.After separating an intact yolk from egg white using an egg separator,Thermo Scientific Pierce Delipidation Reagent is added toseparate the proteins from lipid. The delipidation reagent can alsobe used to store an egg yolk in the freezer for up to one year. Afterdelipidation, the protein-containing sample fraction is mixed withThermo Scientific Pierce IgY Precipitation Reagent to create arelatively pure IgY precipitate that is recovered by centrifugation.Routinely, 80-120 mg of high purity (> 85%), intact IgY can beobtained per egg using the Pierce Kit.References1. Cassidy, P.B., et al. (2006). Carcinogenesis. 27, 2538–2549.2. Kamiya, Y., et al. (2005).J. Biol. Chem. 280, 37178–37182.3. Kantardzhieva, A. et al. (2005). Retinal Cell Biol. 46, 2192–2201.Pierce Chicken IgY Purification KitPurifies 100 mg of chicken IgY with higher purity than ever before!Highlights:• More for your money – purifies twice the amount of IgY as theleading competitor’s kit with a lower cost-per-mg of IgY purified• Higher purity – 85–95% by SDS-PAGE analysis (see Figure)• Ease-of-use – the simple precipitation method works withoutaffinity columns• Flexibility – eggs can be stored in buffer and purified at a later date• Convenience – use eggs directly out of the refrigerator; no needto wait for them to warm upOrdering InformationProduct # Description Pkg. Size44918 Chicken IgY Purification KitSufficient reagents to purify 5 egg yolks.Includes: Pierce Delipidation ReagentPierce IgY Precipitation ReagentPierce Egg Separator44922 Chicken IgY Purification KitSufficient reagents to purify 25 egg yolks.Kit500 ml500 ml121055 Delipidation Reagent 500 ml21057 IgY Precipitation Reagent 500 ml21060 Egg Separator 1SDS-PAGE analysis of Thermo Scientific Pierce Chicken IgY purification.IgY ControlSupplier PPierce KitMW MarkerskDa15010075503525Kit15Chicken IgY was purified according to each manufacturer’s instructions.The gel shows the analysis of 2 µg of protein applied per well. The Pierce IgYKit purified the chicken IgY to a purity level of > 85% using Thermo ScientificGelCode Blue Stain Reagent (Product # 24590). The competitor’s productachieved only a 53% purity level. The arrow indicates intact IgY.52For more information, or to download product instructions, visit www.thermo.com/pierce

Affinity Purification of Specific AntibodiesAlthough Proteins A, G, A/G and L are excellent ligands forpurification of total IgG from a sample, purification of an antibodyspecific for a particular antigen and free of contamination fromother immunoglobulins is often required. This can be accomplishedby immobilizing the particular antigen used for immunization sothat only those antibodies that bind specifically to the antigen arepurified in the procedure. Activated affinity supports that can beused to immobilize peptides or other antigens for use in affinitypurification are described later on pages 10–25.Successful affinity purification of antibody depends on effectivepresentation of the relevant epitopes on the antigen to bindingsites of the antibody. If the antigen is small and immobilizeddirectly to a solid support surface by multiple chemical bonds,important epitopes may be blocked or sterically hindered, prohibitingeffective antibody binding. Therefore, it is best to immobilizeantigens using a unique functional group (e.g., sulfhydryl on asingle terminal cysteine in a peptide) and to use an activatedsupport whose reactive groups occur on spacer arms that areseveral atoms long. For larger antigens, especially those withmultiple sites of immobilization, the spacer arm length becomesless important since the antigen itself serves as an effectivespacer between the support matrix and the epitope. Generally,if the antigen was crosslinked to a carrier protein to facilitateantibody production, best results are obtained when the antigenis immobilized for affinity purification using the same chemistry(e.g., reaction to primary amines, sulfhydryls, carboxylic acids oraldehydes). In this way, all epitopes will be available for antibodybinding, allowing greater efficiency in purification and recovery ofthe specific immunoglobulin.Little variation exists among typical binding and elution conditionsfor affinity purification of antibodies because at the core of eachprocedure is the affinity of an antibody for its respective antigen.Since antibodies are designed to recognize and bind antigenstightly under physiologic conditions, most affinity purificationprocedures use binding conditions that mimic physiologic pH andionic strength. The most common binding buffers are phosphatebuffered saline (PBS) and Tris buffered saline (TBS) at pH 7.2 and1.5 M NaCl (see pages 4–5 for convenient premixed buffer packs).Once the antibody has been bound to an immobilized antigen,additional binding buffer is used to wash unbound material fromthe support. To minimize nonspecific binding, many researchersuse wash buffer containing additional salt or detergent to disruptany weak interactions.Specific, purified antibodies are eluted from an affinity resin byaltering the pH or ionic strength of the buffer (see page 3 forrecipes of common elution buffers). Antibodies generally areresilient proteins that tolerate a range of pH from 2.5 to 11.5 withminimal loss of activity, and pH-shift is by far the most commonelution strategy. In some cases an antibody-antigen interaction isnot efficiently disrupted by pH changes or is damaged by the pH,requiring that an alternate strategy be employed.See section on Covalent Coupling of Affinity Ligands to Solid Supports (page 10) formore information on immobilization of antigens.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.53

Immunoprecipitation and Co-Immunoprecipitation AssaysCo-ImmunoprecipitationImmunoprecipitation can be extended to yet another level ofaffinity purification. In co-immunoprecipitation (co-IP), an antibodyis used to capture not only a specific antigen, but also whateverprotein or complex is bound to the antigen. When Protein A orProtein G is used to affinity-purify the antibody:antigen complex,there is a minimum of three levels of affinity binding interactioninvolved in co-IP. For a co-IP to be successful, the binding bufferconditions must be compatible with all levels of binding interactioninvolved, and the epitope on the antigen must not be blocked byprotein:protein interactions.ImmunoprecipitationImmunoprecipitation (IP) refers to the small-scale affinitypurification of antigen using a specific antibody. Classicalimmunoprecipitation involves the following steps:The complexity of a co-IP system can be reduced by immobilizingthe antibody directly and covalently to a resin. This simplifies thebinding conditions but, more importantly, it allows the precipitatedcomplex to be eluted from the resin while the antibody remainsintact and immobilized. When the precipitated complex isanalyzed by SDS-PAGE or Western blotting, it is free frominterfering antibody heavy- and light-chain and the analysis isgreatly simplified.1. Incubate specific antibody with an antigen-containing sample.2. Capture antibody-antigen complex with Protein A or Protein Gagarose resin (Protein A or Protein G binds the antibody, whichis bound to its antigen).3. Wash the resin with buffer to remove non-bound samplecomponents.4. Elute the antigen (and antibody) by boiling the resin in reducingSDS-PAGE sample buffer.5. Analyze eluted sample by gel electrophoresis.Classical IP is usually performed in a microcentrifuge tube with0.1–1 ml of an antigen-containing sample using 10–50 µl of Protein Aor Protein G agarose. The beaded resin is pelleted by centrifugationafter each step (washes and elution) and the supernatant isremoved. It is practically impossible to identify an elution bufferthat will elute antigen from the antibody without also eluting theantibody from Protein A or Protein G. Therefore, the eluted samplewill always contain both antigen and antibody, and reducing gelelectrophoresis of the eluted sample will yield both the antigenband and heavy- and light-chain antibody fragment bands.To avoid antibody contamination of the eluted antigen, modificationsto the classical IP can be made so that the antibody is permanentlyimmobilized and will not elute with the antigen. One strategy is to firstbind the antibody to the Protein A or Protein G resin and then covalentlycrosslink the antibody to the Protein A or Protein G. Seize XImmunoprecipitation Kits use this approach. Another strategy is todirectly couple antibody to an activated affinity support such asAminoLink Plus Coupling Resin (see pages 11 and 14). Seize PrimaryImmunoprecipitation Kits use this approach. Also, because ThermoScientific Pierce IP Kits use our Spin Cup Columns, they improvereproducibility of IP experiments by eliminating resin loss during thewashing procedures.54For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific Products for Immunoprecipitationand Co-Immunoprecipitation AssaysPierce Classic Immunoprecipitation KitsFor fast, easy recovery of immune complexed proteins.Highlights:• Improve assay consistency – our Spin Cups eliminate resin loss• Easy protocol – immunoprecipitate (IP) out the target proteinfrom crude lysate in just four easy steps• Fast – IP a protein using Pierce Spin Cups and tubes in less thanone hour• Economical – reuse the Immobilized Protein A or Protein Gsupport for future IPs• Complete kits – choose kits with or without cell lysis reagentsfor bacterial, mammalian or yeast cellsStep 1. Incubate antibodywith cell lysateStep 2. Apply sample toProtein A or G Agarose ResinAgaroseBeadProteinGCapture Antibody-Antigen Complexeswith Protein A or G Agarose BeadsHow it worksOur Classic Immunoprecipitation Kits use our Spin Cups andMicrocentrifuge Collection Tubes for easy separation of the solidProtein A or Protein G support from the recovered protein. Thereis no need to carefully pipette supernatant away from the support.Add the sample containing the immune complex to the Protein A orG support and centrifuge to remove nonbound materials. Recoverthe immunoprecipitated protein by adding elution buffer and thenspinning. Next, mix the eluted protein with the sample loadingbuffer supplied in the kit and analyze by SDS-PAGE.Form Immune ComplexStep 3. Centrifuge to remove nonbound proteinsAdd Wash BufferAdd Elution BufferCentrifuge1 minuteStep 4. Elute bound proteins (immune complex)Centrifuge1 minuteStep 5. Analyze proteins on Western blotNonbound ProteinsImmunoprecipitated ProteinThermo Scientific Pierce Spin Cupand Microcentrifuge TubeOrdering InformationProduct # Description Pkg. Size45213 Classic Protein A IP KitSufficient reagent for 50 immunoprecipitations.Includes: Protein A Plus AgaroseSample Loading BufferBinding BufferElution BufferPierce Spin CupsPierce Microcentrifuge Tubes45218 Classic Protein G IP KitSufficient reagent for 50 immunoprecipitations.Includes: Protein G Plus AgaroseSample Loading BufferBinding BufferElution BufferPierce Spin CupsPierce Microcentrifuge Tubes45217 Classic Mammalian IP KitSufficient reagent for 50 immunoprecipitations.Includes: Protein G Plus AgaroseSample Loading BufferBinding BufferElution BufferPierce Spin CupsPierce Microcentrifuge TubesM-PER Mammalian ProteinExtraction ReagentKit1 ml5 ml500 ml50 ml12/pkg.72/pkg.Kit1 ml5 ml500 ml250 ml12/pkg.72/pkg.Kit1 ml5 ml500 ml250 ml12/pkg.72/pkg.25 ml69702 Spin Cups – Cellulose Acetate Filter 50 units69720 Microcentrifuge Tubes, 2 ml 72 tubesMWMarkerLysateThe Thermo Scientific Pierce Classic Immunoprecipitation Kit Protocol.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.55

Thermo Scientific Products for Immunoprecipitationand Co-Immunoprecipitation AssaysSeize X Immunoprecipitation KitsRecover more protein without antibody interference!Seize X Kits extend the functionality of traditional immunoprecipitation(IP) methods by adding crosslinking technology and microcentrifugespin cup sample handling to the procedure. The primary benefitsresulting from these features are the ability to purify target proteinwithout contamination by the antibody and the ability to more effectivelywash and separate samples from the beaded agarose resin.AgaroseBeadProteinGImmobilized Protein GBinds AntibodyDSSCrosslinkerDisuccinimidyl SuberateCrosslinks ProteinsAgaroseBeadProteinGOriented and CovalentlyImmobilized AntibodyHighlights:• No antibody contamination – no elution of antibody heavy and lightchains to interfere in SDS-PAGE analysis of purified protein• Improved assay reliability and sample handling – our Spin Cupseliminate resin loss and provide for more efficient separation ofsolutions compared to traditional IP• Potentially more economical – the prepared antibody affinity resincan be reused multiple times to immunoprecipitate a greater totalamount of protein than in traditional IP• Complete kits – package includes sufficient reagents and spincups to immobilize at least four different antibody samples, eachof which may be reused multiple times for a total of about forty (40)IP experiments123Thermo Scientific Seize X IP Kits purify antigen without interference by the IPantibody. E. coli cells expressing fusion of green fluorescent protein (GFP) wereextracted with B-PER Reagent † (Product # 78248) and then immunoprecipitatedusing a polyclonal goat anti-GFP antibody. Eluted IP products were separatedby SDS-PAGE and coomassie stained (Product # 24590) to detect total protein.Lane 1: single product obtained using the Seize X Protein G IP Kit. Lane 2: antigenand antibody fragments resulting from traditional IP method. Lane 3: molecularweight marker.kDa2151208460392818.3How it worksThe Seize X IP method involves capturing the IP antibody toProtein A or Protein G beaded agarose resin and covalentlyimmobilizing it to the support by crosslinking with ourdisuccinmidyl suberate (DSS) reagent (see Figure). The antibodyresin is then incubated with the sample that contains the proteinantigen of interest, allowing the antibody:antigen complex to form.After washing to remove nonbound sample components, theantigen is recovered using the elution buffer supplied in the kit.The entire procedure is performed in a microcentrifuge spin cup,allowing solutions to be fully separated from the agarose resinupon brief centrifugation. Only antigen is eluted by the procedure(see Figure), enabling it to be identified and further analyzed withoutinterference from antibody fragments. Furthermore, the antibodyresin can be reused for additional rounds of immunoprecipitation.ReferencesIkemoto, A., et al. (2003). J. Biol. Chem. 278, 5929–5940.Kerkela, R., et al. (2002). J. Biol. Chem. 277, 13752–13760.Parkin, S.E., et al. (2002). J. Biol. Chem. 277, 23563–23572.Quill, T.A., et al. (2001). Proc Nat. Acad. Sci. USA 98, 12527–12531.Roti, E.C., et al. (2002). J. Biol. Chem. 277, 47779–47785.Sklyarova, T., et al. (2002). J. Biol. Chem. 277, 39840–39849.Ordering InformationProduct # Description Pkg. Size45215 Seize X Protein A Immunoprecipitation KitSufficient reagents to immobilize 4 different antibodiesand perform 40 total IP reactions.Includes: Protein A Plus AgaroseBinding/Wash BufferElution BufferSample Loading BufferDSSPierce Spin CupsPierce Microcentrifuge Tubes45210 Seize X Protein G Immunoprecipitation KitSufficient reagents to immobilize 4 different antibodiesand perform 40 total IP reactions.Includes: Protein G Plus AgaroseBinding/Washer BufferElution BufferSample Loading BufferDSSPierce Spin CupsPierce Microcentrifuge Tubes45225 Seize X Mammalian Immunoprecipitation KitSame scale and components as Product #45210and also includes:M-PER Mammalian ProteinExtraction ReagentKit1 ml500 ml50 ml5 ml8 x 2 mg12/pkg.72/pkg.Kit1 ml500 ml50 ml5 ml8 x 2 mg12/pkg.72/pkg.Kit25 ml69702 Pierce Spin Cups – Cellulose Acetate Filter 50 units69720 Pierce Microcentrifuge Tubes, 2 ml 72 tubes† See patent information on inside back cover.56For more information, or to download product instructions, visit www.thermo.com/pierce

Seize Primary Immunoprecipitation KitsNo species or subclass requirements and no antibody band interference.Seize Primary Immunoprecipitation Kits eliminate the need todetermine if an antibody species or subclass binds well to ProteinA or Protein G. Using this kit, directly couple a primary antibodyto the AminoLink Plus Activated Support to create a reusable,immunoprecipitation (IP) resin that prevents the antibody fromco-eluting with the target protein.Highlights:• No antibody contamination – antibody heavy and light chainsdo not elute with purified protein; eliminates interference fromantibody heavy and light chains in SDS-PAGE or Western blots• IP with any species and subclass of antibody – use chicken IgY,human IgE, mouse IgM or any purified, carrier-free protein• Prepared antibody affinity resin is reusable for multiple roundsof IP – represents a possible savings of expensive antibody• Convenient sample handling – our Spin Cups eliminate resinloss and provide for efficient separation of solutions• Highest possible yield – retains antibody function better thanother covalent attachment methods (e.g., Seize X Kits)• Complete kits – package includes sufficient reagents and spincups to immobilize at least four different antibody samples, eachof which may be reused multiple times for multiple IP experimentsPercent Coupled10080GoatHumanRabbit 160Rabbit 2MouseChicken400 1 2 3 45Time (Hours)Antibody coupling efficiency of mammalian and avian antibody. Purifiedantibody (200 µg) from various species was coupled to 200 µl of ThermoScientific AminoLink Plus Coupling Resin at 1-hour intervals for 4 hours atroom temperature. For the chicken antibody, 500 µg was used.LysateFlow-throughWash 1Wash 2Wash 3Elution 1Elution 2Elution 3Elution 4MW MarkerAgaroseBeadOCHThermo ScientificAminoLink Plus Resin(Aldehyde Activated)+H 2 NH 2 NNH 2NH 2Antibody withPrimary AminesYYYYYBeadYYCovalentlyImmobilized AntibodyHow it worksBoth Seize Primary and Seize X IP Kits offer a reusable antibodycoupledresin resulting in no antibody heavy and light chain contamination.However, in the Seize X IP Procedure, DSS is used tocrosslink the antibody to the Protein A or Protein G resin. Becausecrosslinking often reduces the antibody-binding capacity, this stephas been eliminated in the Primary IP Kit to allow for greater recoveryof the target protein. While the Classic Kit (page 55) method potentiallyyields a greater quantity of target protein, the presence of antibodyheavy and light chains in the eluent can distort or hide the recoveredtarget protein band on a polyacrylamide gel.ReferencesKwon, Y.H., et al. (2002). J. Biol. Chem. 277, 41417–41422.Eberhardt, W., et al. (2002). Mol. Endocrinol. 16, 1752–1766.Ordering InformationProduct # Description Pkg. Size45335 Seize Primary Immunoprecipitation KitSufficient reagents to immobilize 10 different antibodiesand perform 20 total IP reactions.*Includes: AminoLink Plus Coupling ResinCoupling BufferQuenching BufferWash BufferReducing AgentPierce Microcentrifuge TubesPierce Spin CupsBinding BufferImmunoprecipitation Sample BufferElution BufferLane MarkerSample Buffer, Non-reducingKit2 ml500 ml60 ml60 ml0.5 ml150 each12/pkg.500 ml500 ml50 ml5 ml45332 Seize Primary MammalianImmunoprecipitation KitSame scale and components as Product #45335and also includes:M-PER Mammalian ProteinExtraction ReagentKit25 ml69702 Pierce Spin Cups – Cellulose Acetate Filter 50 units69720 Pierce Microcentrifuge Tubes, 2 ml 72 tubes* Based on a scale of 100–200 µl of settled resin per IP reaction. Kit can provide upto 200 IP reactions if more Pierce Spin Cups are purchased separately.The Thermo Scientific Seize Primary Kit produces exceptionally clean IP results.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.57

Thermo Scientific Products for Immunoprecipitationand Co-Immunoprecipitation AssaysSeize Coated Plate Immunoprecipitation KitsPre-coated 96-well plates are easier to use and faster thantraditional microcentrifuge tube methods.Thermo Scientific Seize Coated Plate IP Kits provide for rapidimmunoprecipitation of multiple samples without the usual tediumof pipetting, centrifuging and separating beaded affinity resin inindividual microcentrifuge tubes. Immunoprecipitation is accomplishedusing coated 96-well microplates rather than beadedagarose resin. The plate format allows for faster processing ofmultiple samples. Select from Protein A/G-, Protein G- or streptavidin-coatedplates.Highlights:• Ready-to-use, high quality coated plates provide high capacityand consistency• Plate format best suited for simultaneously processing multiplesamples and their control conditions• Faster, easier and more thorough washing than with traditionaltube/resin IP methods• Uses familiar and convenient ELISA tools (multichannel pipettorsand plate washing); no tedious separation of supernatant frompelleted resin beads, and no tubes to open and close andcentrifuge• Coated plates are 96-well strip plates, convenient for experimentsrequiring only a partial plate• Easy-to-follow instructions, including detailed explanation ofappropriate controls• Three kits available, suitable for most common antibody types(mouse, rabbit, human and goat IgG subclasses) or anybiotinylated antibody or “bait” protein123 4 5Thermo Scientific Seize Protein GCoated Plate IP Kit (Product # 45355)immunoprecipitation of CD71 (transferringreceptor) from human serumusing and a goat anti-CD71 polyclonalantibody. Eluted products forthe experimental and control sampleswere mixed with nonreducing sampleloading buffer, separated by SDS-PAGE, transferred to nitrocellulosemembrane and detected by Westernblotting with the IP antibody, Goatanti-mouse-HRPconjugated secondaryantibody (Product # 31432) andThermo Scientific SuperSignal WestDura Chemiluminescent Substrate(Product # 34076).Choosing between Protein G, Protein A/G and StreptavidinCoated Plate KitsStreptavidin is a protein that binds specifically and very stronglyto biotin; therefore, the Streptavidin Coated Plate IP Kit (#45360) isappropriate for immunoprecipitation when using a biotin-labeled(biotinylated) antibody. In fact, this kit can be used to affinity-purifya binding partner to any antibody species or subclass or any otherprotein or molecule that is biotinylated. Because the streptavidinbiotinaffinity interaction is so strong, the elution step generally willdissociate only the antigen (binding partner), not the biotinylatedantibody or “bait” protein. See page 26 for more information aboutavidin:biotin binding.Protein A and Protein G are different proteins that bind to immunoglobulins(primarily only IgG). Typically, Protein A is preferred foruse with Rabbit polyclonal antibodies, while Protein G is preferredfor use with mouse antibodies (especially monoclonals of the IgG 1subclass). Protein A/G is a recombinant of Protein A and Protein Gthat has the additive binding properties of both proteins. Seepage 37 for more information about Protein A, G and A/G.ReferenceDesai, S. and Hermanson, G. (1997). Previews 1(3), 2–7.Ordering InformationProduct # Description Pkg. Size45350 Seize Protein A/G Coated Plate IP KitAntibody binding capacity/well: 2.5 µg.Sufficient capacity for downstream analysisof IP or co-IP proteins in-gel or by Western blot.Includes: Protein A/G Coated 12 x 8-Well Strip PlatesPhosphate Buffered SalineSurfact-Amps X-100 (10% Triton X-100)Elution BufferNeutralization BufferUncoated 96-well Strip Plates (white),(for sample collection and neutralization)Plate Sealers45360 Seize Streptavidin Coated Plate IP KitAntibody binding capacity/well: 5 µg.Sufficient capacity for downstream analysisof IP or co-IP proteins in-gel or by Western blot.Includes: High Binding Capacity StreptavidinCoated PlatesBiotin Blocking BufferPhosphate Buffered SalineSurfact-Amps X-100 (10% Triton X-100)Elution BufferNeutralization BufferUncoated 96-well Strip Plates (white),(for sample collection and neutralization)Plate SealersKit2 plates2 packs6 x 10 ml50 ml7 ml2 each18 sheetsKit2 plates30 ml2 packs6 x 10 ml50 ml7 ml2 each18 sheetsLane 1: Experiment (immunoprecipitation product)Lane 2: Antibody-only control (no sample)Lane 3: Human serum sample control (no antibody)Lane 4: Plate control (no antibody or human sample)Lane 5: Pure target protein (CD71) for size reference58For more information, or to download product instructions, visit www.thermo.com/pierce

Pierce Co-Immunoprecipitation KitsAll the components needed to perform a properly controlledco-IP experiment.Co-immunoprecipitation, or co-IP as it is widely known, is thegateway method to all other in vitro methods for protein interactionanalysis. As a result, co-IP is among the most common techniquesfor protein:protein interaction discovery and verification. Forexample, protein interactions discovered through the use of yeasttwo-hybrid experiments are most often confirmed by co-IPexperiments. The Thermo Scientific Pierce Co-ImmunoprecipitationKits were configured to provide the essential tools to effectivelyperform a co-IP experiment using the primary antibody of yourchoice. These kits provide those attempting a co-IP for the firsttime, as well as those experienced in the method, an idealsystem for designing, constructing and performing a controlled co-IP experiment.Benefits of target antibody immobilizationOur Co-Immunoprecipitation Kit utilizes Thermo ScientificAminoLink Plus Coupling Resin, which brings several benefits tothe co-IP application:• The AminoLink Coupling Resin allows a purified antibody tobe directly and covalently immobilized onto the matrix whileretaining antibody activity• The antibody-coupled support retains the antibody duringelution of the co-IP complex, simplifying analysis• Detection of the co-precipitated products by SDS-PAGE isaccomplished without interference from antibody fragments• Covalent attachment of antibody conserves costly antibodyand allows the matrix to be used repeatedlyLabeled ProteinsMDM2p53LucMDM2+p53CoIPMDM2+Luc— MDM2— Luciferase— p53Highlights:• AminoLink Plus Coupling Resin for covalent attachment ofprimary antibody prevents co-elution of antibody or antibodyfragments with the target protein interactors• Couple any purified antibody regardless of species or Ig subclass(chicken IgY, human IgE, mouse IgG 1 , IgM, etc.)• Physiologic co-IP buffer for optimal binding• Control gel included – allows for a properly controlled co-IPexperiment• Spin cup columns for efficient washing and elution of smallgel volumes• Coupled antibody support reusable up to 10 times – savesprecious antibody.Ordering InformationProduct # Description Pkg. Size23600 Pierce Co-IP KitSufficient material to immobilize up to 10 antibodiesand perform a minimum of 40 co-IP reactions if25 µl of antibody immobilized support is used.More co-IPs can be performed if smaller volumesare used.Includes: AminoLink Plus Coupling Resin(4 ml of a 50% slurry)Control Resin, (4 ml of a 50% slurry)BupH Modified Dulbecco’s PBS,(Coupling Buffer and Co-IP Buffer)(1 L total volume)Quenching BufferWash SolutionSodium Cyanoborohydride Solution (5 M)Pierce Spin CupsPierce Microcentrifuge Tubes, 1.5 mlIgG Elution BufferLane MarkerSample Buffer, Non-reducing (5X)23605 Pierce Mammalian Co-IP KitSufficient material to immobilize up to 10 antibodiesand perform a minimum of 40 co-IP reactions if 25 µlof antibody immobilized support is used. Moreco-IPs can be performed if smaller volumes are used.Includes same components as Product # 23600and also includes:M-PER Lysis BufferKit2 ml2 ml2 packs60 ml60 ml0.5 ml50 ea.144 ea.50 ml5 mlKit25 mlCo-IP of p53 and MDM2 with mouse MAb to MDM2 using components of theThermo Scientific Pierce Co-Immunoprecipitation Kit. Purified mouse anti-MDM2 was coupled to Thermo Scientific AminoLink Plus Coupling Resin.Luciferase, MDM2 and p53 were translated and 35 S-labeled. In vitro translatedp53 (5 µl) and MDM2 (5 µl) were combined and incubated at 30°C for 30 minutes.Co-IP was performed at 4°C for 2 hours with 60 µl anti-MDM2 antibodycoupledresin. Luciferase was used as a negative control protein to incubatewith MDM2. Eluted proteins were resolved by 4–20% SDS-PAGE and visualizedby autoradiography.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.59

Thermo Scientific Products for Immunoprecipitationand Co-Immunoprecipitation AssaysPierce HA and c-Myc IP/Co-IP KitsConfirm two-hybrid interactions.The Thermo Scientific Pierce HA and c-Myc Tagged Protein Co-IPKits include high-affinity, high-specificity antibodies coupled toagarose to enable capture of HA- or c-Myc-tagged proteins alongwith their binding partners. The covalent linkage between antibodyand the resin ensures results free from antibody contaminationand with minimal background. The mammalian kits also includeM-PER Mammalian Protein Extraction Reagent, which quickly andgently lyses mammalian cells for easy preparation of extracts thatare compatible with co-immunoprecipitation (co-IP).Highlights:• Specific, high-affinity antibody provides high yield andminimal background• No antibody contamination in eluted sample• Control agarose resin included to test nonspecific binding orpre-clear sample• Control tagged protein included to verify kit performance• Complete kits for IP or co-IP, with no reagent formulation necessary• Scalable for different antibody-resin and lysate requirements• Includes Mini-Spin Columns for efficient washing and elution stepsGST-HA —MW MarkerHA LysateIPSupplier ASupplier BSupplier CThermo Scientific PierceMW Markerc-Myc LysateIPSupplier ASupplier BSupplier CSupplier DThermo Scientific Pierce— GST-c-MycComparison of the effectiveness of anti-HA- and anti-c-Myc-coupled resins.Thermo Scientific Pierce Anti-HA and anti-c-Myc resins show high affinity andhigh specificity. Each IP was conducted with the same amount of antibody(10 µg of immobilized HA antibody or 5 µg of immobilized c-Myc antibody) andthe same amount of GST-HA or GST-c-Myc tag containing lysate (50 µl).LTA Lysatec-Myc-p53 LysateCo-IPIPNeg. Control1 2 3 4 5 6— LTA— c-Myc-p53Co-IP of SV40 large T-antigen (LTA) with c-Myc-tagged p53. LTA andc-Myc-p53 were expressed 35 S-labeled in vitro (Lane 1 and 2). Before IPand co-IP, the lysates (5 µl of each) of LTA and c-Myc-p53 (Lanes 3 and 6),c-Myc-p53 alone (Lane 4) or LTA alone (Lane 5) were incubated at 30˚C for1 hour. Anti-c-Myc agarose (5 µg antibody in 10 µl slurry) (Lanes 3, 4 and 5) orplain agarose slurry (Lane 6) was added to the corresponding sample. IP andco-IP reactions were performed at 4˚C overnight. IP and co-IP products wereeluted and separated by 12% SDS-PAGE. The 35 S-labeled proteins weredetected by fluorography.Ordering InformationProduct # Description Pkg. Size23610 Pierce HA-Tag IP/Co-IP KitSufficient materials for 25 IP/co-IP assays with HAtaggedproteins.Includes: Anti-HA Antibody Agarose ResinTris Buffered Saline PackElution Buffer, pH 2.8Sample Loading Buffer (5X)Pierce Spin CupsPierce Microcentrifuge TubesHA-Tagged Positive Control23615 Pierce Mammalian HA-Tag IP/Co-IP KitSame scale and components as Product #23610and also includes:M-PER Mammalian ProteinExtraction Reagent23620 Pierce c-Myc-Tag IP/Co-IP KitSufficient materials for 25 IP/co-IP assays withc-Myc-tagged proteins.Includes: Anti-HA Antibody Agarose ResinTris Buffered Saline PackElution Buffer, pH 2.8Sample Loading Buffer (5X)Pierce Spin CupsPierce Microcentrifuge Tubesc-Myc-Tagged Positive Control23625 Pierce c-Myc-Tag IP/Co-IP KitSame scale and components as Product #23620and also includes:M-PER Mammalian ProteinExtraction ReagenttNeg. ControlKit150 µl1 pack50 ml5 ml27/pkg100/pkg500 µlKit25 mlKit250 µl1 pack50 ml5 ml27/pkg100/pkg500 µlKit25 ml60For more information, or to download product instructions, visit www.thermo.com/pierce

Pierce Plus IgG Orientation KitsGreater antibody-binding capacities than Classic IgG OrientationKits; available with either recombinant Protein A or Protein G.Thermo Scientific Pierce Plus IgG Orientation Kits provide forefficient binding and covalent crosslinking of antibodies to ThermoScientific Pierce Protein A and Protein G Agarose Resin for usein affinity column purification of antigens. The method improvesupon the traditional orientation mechanism by replacing the use ofdimethyl pimelimidate (DMP) with disuccinimidyl suberate (DSS)for the crosslinking step. Our high-quality Protein A and ProteinG Agarose Resin provides for efficient antibody binding andchromatography, and the DSS provides for simple and efficientcrosslinking of the bound antibody. The result is an affinity columnthat can be used multiple times to purify the specific antigen of theimmobilized antibody from cell lysates and other samples.Highlights:• DSS crosslinking system allows for higher antibody loading andless antibody leaching than imidoesters like DMP• Allows the positioning of IgG with the antigen-binding sitesdirected outward, capturing passing antigen in the mobile phase• Complete kits include all the reagents needed to immobilizethe antibodyThermo Scientific Pierce Protein G IgG Plus Orientation kit schematicAgaroseBeadProteinGDSSCrosslinkerReferencesVenturi, M., et al. (2000). J. Biol. Chem. 275(7), 4734–4742.Sharp, D.A., et al. (2005). J. Biol. Chem. 280, 19401–19409.Liang, T.W., et al. (2002). J. Immunol. 168, 1618–1626.Ordering InformationAgaroseBeadProteinGProduct # Description Pkg. Size44893 Pierce Protein A IgG Plus Orientation KitSufficient reagents for preparing 2 x 2 ml columns.Maximum recommended loading capacity:16 mg Rabbit IgG per columnIncludes: Recombinant Protein A Agarose ColumnsDSS CrosslinkerPhosphate Buffered SalineBlocking BufferElution BufferBinding/Wash BufferColumn Accessories44990 Pierce Protein G IgG Plus Orientation KitSufficient reagents for preparing 2 x 2 ml columns.Maximum recommended loading capacity:16 mg Rabbit IgG per columnIncludes: Recombinant Protein G Agarose ColumnsDSS CrosslinkerPhosphate Buffered SalineBlocking BufferElution BufferBinding/Wash BufferColumn AccessoriesKit2 x 2 ml2 x 13 mg1/pkg6 ml15 ml120 mlKit2 x 2 ml2 x 13 mg1/pkg6 ml15 ml120 mlTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.61

Affinity Based Contaminant RemovalRemoval of DetergentHowever necessary the use of detergent may have been for initialcell lysis or membrane protein extractions, subsequent applicationsor experiments with the extracted proteins often require removalof some or all of the detergent. For example, although many watersolubleproteins are functional in detergent-solubilized form,membrane proteins are often modified and inactivated by detergentsolubilization as a result of native lipid interactions having beendisrupted. In some such cases, membrane protein function isrestored when they are reconstituted into bilayer membranes byreplacement of detergent with phospholipids or other membranelikelipid mixtures.Contaminant RemovalFollowing the primary purification procedure to obtain a sampleof interest, secondary purification steps to remove contaminantsmay be required. The contaminants might be inhibitors, interferingsubstances or inappropriate buffers. A number of available affinitysupports allow researchers to either specifically purify a proteinof interest away from a complex mixture of biological molecules(positive selection) or remove specific contaminants from a samplecontaining a protein of interest (negative selection). Nearly anygiven affinity purification system can be used for either positive ornegative selection, depending on whether the non-bound or elutedfraction is recovered. For example, immobilized Protein A can beused for general affinity purification of antibodies (positive selection),but it can also be used to selectively remove immunoglobulins froma sample in which they are considered a contaminant (negativeselection). Following is a brief description of affinity-basedsystems for removal of contaminants by negative selection.The function of an individual protein can be studied in isolation if itis first purified and then reconstituted into an artificial membrane(although recovery of native orientation in the membrane is amajor challenge). Even when restoration of protein function isnot an issue, detergent concentration might have to be decreasedin a sample to make it compatible with protein assays or gelelectrophoresis.Detergent removal can be attempted in a number of ways.Dialysis is effective for removal of detergents that have high CMCsand/or small aggregation numbers, such the N-octyl glucosides.Detergents with low CMCs and large aggregation numberscannot be dialyzed because most of the detergent molecules existin micelles that are too large to diffuse through the pores of thedialysis membrane; only excess monomer can be dialyzed. Ionexchange chromatography using appropriate conditions toselectively bind and elute the proteins of interest is anothereffective way to remove detergent. Sucrose density gradientseparation also can be used.Thermo Scientific Detergent Removal Gel allows selectiveaffinity-based removal of many different detergents from solutions.62For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific Products for Affinity BasedContaminant RemovalDetergent Removing GelMakes detergent removal efficient, fast and easy with high proteinrecoveries, too.Highlights:• Allows relatively small detergent molecules to enter the gelmatrix where they interact with a specially developed ligandcapable of removing them from solution• Low exclusion limit of the support overcomes nonspecific binding• For use with biological macromolecules greater than 10 kDa• Detergent is extracted without loss of valuable protein• Reusable affinity matrix can be regenerated up to three times• Compatible with a wide variety of buffers, pH 3.5–10• Recovery of dilute protein solutions enhanced by use of acarrier proteinApplications:• Reconstitution of proteoliposomes to study integral membraneproteins 1,2• Preparation of samples for mass spectroscopy 3,4Detergent binding data.Detergent Product #Capacity(mg/ml gel)Binding ConditionsBrij ® -35 28316 80 100 mM Phosphate Buffer, pH 7.0CHAPS 28300 50 50 mM Tris Buffer, pH 9.0SDS 28312 92 50 mM Tris Buffer, pH 9.0Triton ® X-100 28314 57 100 mM Phosphate Buffer, pH 7.0Tween ® -20 28320 74 100 mM Phosphate Buffer, pH 7.0References1. Bubien, J.K., et al. (2001). J. Biol. Chem. 276, 8557–8566.2. Carman, C.V., et al. (2000). J. Biol. Chem. 275, 10443–10452.3. Rouse J.C. and Vath J.E. (1996). Anal Biochem. 238, 82–92.4. Gentile, F., et al. (1997). J. Biol. Chem. 272, 639–646.Ordering InformationProduct # Description Pkg. Size20208 Detergent Removing Gel 10 ml20303 Detergent Removing Gel 10 x 10 ml20346 Detergent Removing Gel 5 x 1 mlpre-packedcolumnsTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.63

Thermo Scientific Products for Affinity BasedContaminant RemovalRemoval of AlbuminHuman serum albumin (HSA) can account for greater than 60% ofthe total protein in serum samples and can have a concentrationof ~40 mg/ml. This high concentration of albumin frequently interfereswith analysis of proteins of biological interest. Traditionally,researchers have produced albumin-free samples using chromatographicmethods involving multiple purification steps. The ThermoScientific Pierce Blue Albumin Removal Kit takes advantage of thealbumin-binding properties of immobilized Cibacron ® Blue F3GADye and is designed for rapid treatment of small sample volumescommonly used in proteomic studies.Pierce Blue Albumin Removal KitAlbumin-free serum samples in less than 15 minutes.The Thermo Scientific Pierce Blue Albumin Removal Kit isdesigned to remove albumin from human serum quickly andconsistently while achieving higher protein yields.Each Blue Disc has the capacity (≥ 2 mg HSA) to remove themajority of albumin from 10–150 µl of serum in less than 15 minutes.The kit procedure has been optimized for removal of humanserum albumin but will also effectively remove swine and sheepserum albumin. With a slight modification of the binding buffer,the Blue Albumin Removal Kit can also be used to remove excessbovine, calf, goat and rat albumin. The product, however, is notrecommended for mouse albumin.AAlbuminContaminationBAlbuminDepletedThermo Scientific Pierce Blue Albumin Removal Kit for 2-D gel analysis.Serum sample obtained by diluting 10 µl human serum with 40 µl TBS (Product# 28376) and loading 5 µl onto Gel A. Albumin-free sample obtained by diluting50 µl human serum 1:1 with buffer, adding to a Pierce Blue Disc, washing theresin three times with 50 µl buffer, combining the fractions, and loading 5 µlonto Gel B. Both samples were focused using pH 4–7 isoelectric focusing (IEF)strips and run on 8–16% Tris-glycine gels.Ordering InformationProduct # Description Pkg. Size89845 Pierce Blue Albumin Removal Kit †Sufficient reagents for 12–25 reactions.Includes: Pierce Blue Albumin Removal DiscsBinding/Wash BufferPierce Spin Columns† See patent information on inside back cover.Kit25 discs6.25 ml25 columnsOur Blue Albumin Removal Buffer is compatible with most downstreamapplications and does not interfere with protein assays orintroduce contaminants for SDS-PAGE. Simply add the albumindepletedserum to the appropriate sample buffer and the sample isready for any application including 2-D gel applications.SERUMFILTRATEBUFFERStep 1.Hydrate Thermo ScientificPierce Disc in water. Spin.Step 2.Add serum sample to the resin.Incubate 2 minutes.Step 3.Recover filtrate and add to the resin.Incubate 2 minutes and spin.Step 4.Add Binding/Elution Buffer.Spin. Wash 1-4 times.Step 5.Combine desired fractions.Concentrate if needed.Total Time: 10-15 minutesThermo Scientific Pierce Albumin Removal Kit protocol.64For more information, or to download product instructions, visit www.thermo.com/pierce

Removal of EndotoxinEndotoxins are pyrogenic lipopolysaccharide (LPS) componentsof gram-negative bacteria. Eliminating endotoxin contaminationin aqueous and physiological solutions is a difficult and oftenexpensive process. Complete removal of all contaminating pyrogensis not feasible; the most likely outcome is a reduction of endotoxinsto tolerable levels for a given study system. Typically, a pyrogenpoor(i.e., safe for use) preparation of protein will contain less than1.0 endotoxin unit (EU) per milligram of protein.Thermo Scientific Detoxi-Gel Endotoxin Removing Gel uses immobilizedpolymixin B to bind and remove endotoxins from solutions.The product is commonly used to remove endotoxins from proteinsolutions, cell culture media, solutions containing pharmacologicalcomponents and aqueous buffers.Certain substances and proteins in solution bind strongly toendotoxin, thereby either decreasing binding capacity of Detoxi-Gel Support or resulting in loss of protein from the sample (i.e.,the protein remains bound to endotoxin when the endotoxin bindsto the immobilized polymixin B). This is especially true of proteinslike bovine serum albumin (BSA) and ovalbumin. BSA is a commoncomponent of tissue media and, as such, it is also often a sourceof endotoxin contamination.Detoxi-Gel Endotoxin Removing GelEliminate worries about endotoxins interfering with your test results.Highlights:• Uses immobilized polymixin B to remove endotoxins by bindingto their lipid A domains• 1 ml of Thermo Scientific Detoxi-Gel Resin removes at least 9,995endotoxin units from a 5 ml sample containing 10,000 EU of lipopolysaccharide(LPS)• Reusable – no loss in binding capacity even after 10 regenerations• Requires activation and regeneration with 1% sodium deoxycholate• Protein recovery dependent upon protein type and concentration– empirical testing requiredReferencesChigaev, A., et al. (2003). J. Biol. Chem. 278, 38174–38182.Gao, B. and Tsan, M. (2003). J. Biol. Chem. 278, 22523–22529.Hahn-Dantona, E., et al. (2001). J. Biol. Chem. 276, 15498–15503.Zhang, J., et al. (2000). FASEB J. 14, 2589–2600.Ordering InformationProduct # Description Pkg. Size20344 Detoxi-Gel Prepacked Columns 5 x 1 ml20339 Detoxi-Gel Endotoxin Removing Gel 10 ml20340 Detoxi-Gel Endotoxin Removing Gel 1,000 mlRemoval of Nonspecific AntibodiesBecause they contain mixtures of immunoglobulins, preparationsof polyclonal antibodies from serum or other samples often havecross-reactivity to unintended targets in the sample being probedin Western blot or ELISA. It is important to purchase or prepareantibodies that do not cross-react in this way. For example, bypassing a rabbit anti-mouse IgG polyclonal antibody sample overa column of immobilized human serum proteins, one can ensurethat the resulting antibody will react only to mouse IgG primaryantibody without cross-reacting with human immunoglobulinsin the sample. We offer selected antibodies that have been preadsorbedin this way to prevent cross-reactivity to unintendedimmunoglobulin species. Such pre-adsorption of antibodies isa form of negative selection affinity purification wherein theimmobilized ligand is a mixture of proteins.Cross-reactivity of antibodies to bacterial proteins is a commonproblem for researchers investigating recombinant proteinsprepared in Escherichia coli. The possibility of such crossreactivitycan be reduced or eliminated by removing immunoglobulinsin the polyclonal antibody sample that bind to proteins fromE. coli. We offer the Thermo Scientific Immobilized E. coli Lysate Kitfor this purpose. Proteins from total lysate of E. coli strain BMH 71–18have been immobilized onto a crosslinked 4% agarose support.Immobilized E. coli Lysate and KitFor clean, easy removal of E. coli-reactive antibodies.High background, or low signal:noise ratio, is often a problemwhen screening libraries. Crude antisera and ascites fluid oftencontain IgG components that bind to Escherichia coli proteins. Thiscan be especially problematic if the titer or binding affinities of theE. coli binding antibodies are higher than that of the antibody to theprotein of interest, resulting in false positives.To make removal of E. coli antibodies cleaner and easier, weimmobilized E. coli proteins on a solid support. When a sample ispassed over the column, the purified antibody is collected in thevoid volume. The contaminating E. coli antibodies are adsorbedonto the matrix, and the purified antisera is collected.Ordering InformationProduct # Description Pkg. Size44938 Immobilized E. coli LysateE. coli proteins from strain BMH 71–18immobilized on crosslinked 4% beaded agarose.44940 Immobilized E. coli Lysate KitIncludes: Prepacked Column of ImmobilizedE. coli LysateBupH Tris Buffered SalineRegeneration BufferColumn Extender5 mlKit2 ml500 ml250 mlTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.65

Additional Thermo Scientific Affinity SupportsImmobilized Soybean Trypsin InhibitorFor effective removal of trypsin, chymotrypsin and elastase fromprotein digests.Applications:• Purifying trypsin, chymotrypsin and elastase 1,2• Removing proteases from activated pancreatic juices 3References1. Feinstein, G., et al. (1974). Euro. J. Biochem. 43(3), 569–581.2. Peterson, L.M., et al. (1976). Biochemistry 15(12), 2501–2508.3. Reeck, G.R., et al. (1971). Biochemistry 10(25), 4690–4698.Ordering InformationIn addition to the few affinity supports whose ligands have broadapplication to many different protein methods, there are manyothers whose applications are more narrowly defined or are incorporatedinto kits for very specific purposes. These include kitsto isolate cell surface proteins using biotinylation and NeutrAvidinAgarose, kits to enrich for phosphoproteins, glycoproteins andubiquitinated proteins. The stand-alone resins on this page and thenext include those that can be used to remove trypsin from proteindigest, isolate blood proteins, purify C-reactive protein and captureribonucleosides. In addition, page 74 presents our complete line ofmagnetic beads, together with their magnet accessories.Product # Description Pkg. Size20235 Immobilized Soybean Trypsin InhibitorSupport: 4% beaded agaroseCapacity: ≥ 6 mg trypsin/ml of resinImmobilized PepstatinAn excellent cathepsin binding matrix.AgaroseBeadHNHNHN2 mlPepstatinPepstatin =RAlaRValVali-ValR = (4-amino-3-hydroxy-6-methyl) heptanoic acidThermo Scientific Immobilized PepstatinReferenceHelseth, Jr., D.L. and Veis, A. (1984). Proc. Natl. Acad. Sci. USA 81, 3302–3306.Ordering InformationProduct # Description Pkg. Size20215 Immobilized PepstatinSupport: Crosslinked 6% beaded agaroseSpacer: DiaminodipropylamineCapacity: 1–2 mg of pepsin/ml of resin5 ml66For more information, or to download product instructions, visit www.thermo.com/pierce

Additional Thermo Scientific Affinity Supports and KitsImmobilized HeparinUse to isolate many blood proteins that have enzymatic activities.Immobilized D-GalactoseFor lectin and galactosidase binding.Applications:• Enrich lysates for nucleic acid-binding proteins• Isolate many blood proteinsReferenceSmith, P.K., et al. (1980). Anal. Biochem. 109, 466–473.Applications:• Human alpha-galactosidase A purification 1• E. coli heat labile enterotoxin purification 2• C-type lectin purification 3• Cholera toxin (CT) purification 4,5AgaroseBeadOCOO -OHOOCH 2 OSO - 3OOHOAgaroseBeadOOSOOHOOOHOHOHOSO 3-NHOSO 3-nThermo Scientific Immobilized Thio-alpha-D-GalactoseOrdering InformationThermo Scientific Immobilized HeparinProduct # Description Pkg. Size20207 Immobilized HeparinSupport: 4% beaded agaroseLoading: ≥ 0.2 mg of heparin/ml of resin(determined by the colorimetric method)Immobilized p-Aminophenyl Phosphoryl CholineFor C-reactive protein binding.KitReferences1. Yasuda, K., et al. (2004). Protein Expression and Purification. 37, 499–506.2. Okamoto, K., et al. (1998). J. Bacteriol. 180, 1368–1374.3. Matsumoto, J., et al. (2001). Development. 128, 3339–3347.4. Bowman, C.C. and Clements, J.D., et al. (2001). Infec. Immun. 69, 1528–1535.5. Tinker, J.K., et al. (2003). Infec. Immun. 71, 4093–4101.Ordering InformationProduct # Description Pkg. Size20372 Immobilized D-GalactoseSupport: 6% beaded agaroseCapacity: ≥ 20 mg jacalin/ml resinImmobilized Boronic Acid5 mlAgaroseBeadHNOOOPO -ON +For ribonucleoside isolation.ResinBeadHNOHBOHThermo Scientific Immobilized p-Aminophenyl Phosphoryl CholineOOHReferenceRobey, F.A. and Liu, T.Y. (1981). J. Biol. Chem. 256, 969–975.Ordering InformationProduct # Description Pkg. Size20307 Immobilized p-Aminophenyl PhosphorylCholineSupport: Crosslinked 6% beaded agaroseCapacity: ≥ 3 mg of human C-reactive protein/ml of resin5 mlThermo Scientific Immobilized Boronic AcidReferencesVlassara, H., et al. (1981). Proc. Natl. Acad. Sci. USA 78, 5190–5192.Gehrke, C.W., et al. (1978). J. Chromatogr. 150, 455–476.Ordering InformationProduct # Description Pkg. Size20244 Immobilized Boronic AcidSupport: Polyacrylamide resin beadsCapacity: ≥ 99% binding and recovery of 110 µmolAMP/ml of resinSpacer: m-aminophenyl groupLoading: 100 µmol boronate/ml of resin10 mlTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.67

Additional Thermo Scientific Affinity Supports and KitsPierce Cell Surface Protein Isolation KitConvenient biotinylation and isolation of cell surface proteins forWestern blot analysis.The Thermo Scientific Cell Surface Protein Isolation Kit is acomplete kit for the convenient biotinylation and isolation ofmammalian cell surface proteins, specifically targeting cellsurface proteins to the exclusion of intracellular proteins.The kit efficiently labels proteins with accessible lysineresidues and sufficient extracellular exposure.Highlights:• Isolates cell surface proteins – reduces complexity of totalcellular protein• Efficiently recovers labeled proteins – cleavable biotin allowsfor nearly 100% recovery of isolated cell surface proteins• Convenience – all reagents are provided in one kit, along withcomplete instructions for labeling, cell lysis and purification ofcell surface proteins• Western blotting applications – proteins recovered in SDS-PAGEbuffer are loaded directly onto polyacrylamide gels• Robust system – protocol designed for diverse cell linesHow it worksThe cell surface protein isolation procedure uses a cell-impermeable,cleavable biotinylation reagent to label surface proteins atexposed primary amines. Cells are then harvested and lysed, andthe labeled surface proteins are affinity-purified using ThermoScientific NeutrAvidin Agarose Resin (see Figure below).The biotinylation reagent, Sulfo-NHS-SS-Biotin, does not permeateintact cell membranes so labeling occurs only on proteins exposedto the cell surface. Labeling occurs at primary amines (-NH 2groups), which exist in proteins at the N-terminus and side chainof lysine residues. After cell lysis, the cell surface proteins thatwere biotinylated by Sulfo-NHS-SS-Biotin are captured by its highaffinityinteraction with NeutrAvidin Agarose Resin. While surfaceprotein are bound to the resin beads, all other proteins and cellularcomponents in the lysate are washed away using the convenientmicrocentrifuge spin columns supplied in the kit. Finally, becauseSulfo-NHS-SS-Biotin contains a disulfide bond (-S-S-) in its spacerarm, the labeled cell surface proteins are efficiently recovered fromthe resin by cleaving the disulfide bond with SDS-PAGE sampleloading buffer that contains dithiothreitol (DTT). The isolated cellsurface proteins contain a small, nonreactive tag of the originallylabeled primary amines but are no longer biotinylated (biotinremains bound to the resin).NBiotinylate cells30 minutes at 4°CQuench ReactionHarvest Cells45403530252015107550Transfer cell pelletto 1.5 ml tubeLyse cells30 minutes on iceIsolate biotinylated proteinson Thermo ScientificNeutrAvidin ResinbeadNBNWash gel then elute with SDS-PAGEsample buffer + 50 mM DTTbead N+protein – SHBSHPerformelectrophoresisor other application1-D gelS-S-proteinNThermo Scientific NeutrAvidinBiotin-Binding ProteinBBiotinProcedure for the Thermo Scientific Pierce Cell Surface Protein Isolation Kit.68For more information, or to download product instructions, visit www.thermo.com/pierce

A. Cell Surface Proteins+———R F E+———R F EEGFREIGF-1Rβ-———R F E-———R F EB. Intracellular Proteins+-——— ———R F E R F E+———R F E+———R F E+———R F EIntegrin α5Integrin β1-———R F E-———R F E-———R F EOrdering InformationProduct # Description Pkg. Size89881 Pierce Cell Surface ProteinIsolation KitIncludes: EZ-Link Sulfo-NHS-SS-BiotinQuenching SolutionLysis BufferNeutrAvidin Agarose ResinWash BufferSpin Columns and AccessoriesDithiothreitol (DTT)Phosphate Buffered SalineTris Buffered SalineKit8 x 12 mg vials16 ml4.5 ml2.25 ml34 ml1 pack8 x 7.7 mg microtubes2 packs1 packHsp90CalnexinProtein isolation is specific to cell surface proteins. Panels are Western blotresults for known cell surface proteins (Panel A) and intracellular proteins(Panel B) from HeLa cells tested with the Thermo Scientific Pierce Cell SurfaceProtein Isolation Kit. Plus symbol (+) denotes results for cells treated with theSulfo-NHS-SS-Biotin reagent; minus symbol (-) denotes results for cells thatwere not treated with the biotin reagent but were otherwise carried throughthe kit procedure. Lanes are no-sample resin-control (R), flow-through (F) andeluted (E) fractions. Presence of target cell surface proteins in the plus-E andminus-F conditions indicate successful isolation with the kit. Presence of intracellularproteins in F condition of both plus and minus conditions indicates thatthe labeling and purification procedure is specific to cell surface proteins.Differential expression of cell surface proteins in response to EGF. A431 andHeLa cells were treated with or without 20 ng/ml and 10 ng/ml EGF for 16 hours,respectively. Both cell types were processed with the Thermo Scientific Pierce CellSurface Protein Isolation Kit protocol. Elution fractions were analyzed by WesternA. A431 CellsB. HeLa CellsEGFIntegrin α5- - + +EGFEGFR- -++- -++Integrin β1blot for the quantities of A. integrin β1 and integrin α5 subunits or B. EGFR.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.69

Additional Thermo Scientific Affinity Supports and KitsPierce Phosphoprotein Enrichment KitFT W E LEnrich phosphoproteins for analysis in Western blotting ormass spectrometry.The Thermo Scientific Pierce Phosphoprotein Enrichment Kit isdesigned for the efficient enrichment of phosphorylated proteinsderived from mammalian cells and tissues. The proprietary resinand buffer composition produces superior yields with negligiblenon-specific binding. The kit is offered in a convenient spin formatand is compatible with downstream applications includingWestern blotting, Mass Spec, 2D-PAGE, and protein arrays.Highlights:• Specific – low nonspecific protein contamination from complexbiological samples, such as cell culture lysate and mousetissue extract• Fast – easy spin format enables enrichment of phosphorylatedproteins in less than 2 hours• Superior Yield – achieves higher yields than other commerciallyavailable kits (see Table)• Convenient format – complete kit offering pre-dispensed spincolumns, buffers, reagents and ultrafiltration columns forenrichment• Compatible – downstream applications include mass spectrometry,Western blotting, and 2D-PAGEThe Thermo Scientific Pierce Phosphoprotein Enrichment Kit provides higherphosphoprotein yields in less time than other kits.KitPhosphoproteinYield (µg) †Enrichment Time(Hours)Our Phosphoprotein Enrichment Kit 300 1.5Supplier Q Kit 88 4.5Supplier I Kit 52 ‡ 3.5Supplier C Kit 160 3Supplier E KitToo dilute todetermine5Phospho-MAPK T202/Y204Phospho-Akt S473Phospho-Src Y527Cytochrome Cp15Ink4bEnrich complex biological samples for phosphorylated proteins with highspecificity. Western blotting analysis reveals a stronger signal for the elutionfraction (E) bands (processed sample) than the total cell extract (L) bands(unprocessed sample) for all phosphoproteins tested. Furthermore, thenon-phosphorylated proteins cytochrome c and p15Ink4b are negative controlsand demonstrate the specificity of the kit. Total cell extract (2 mg) from serumstarvedNIH 3T3 cells was used for each enrichment procedure. Ten µg oftotal protein was loaded in each lane and Western blotting was performedusing antibodies that detect site-specific phosphorylation. FT: flow-throughand W: wash.Ordering InformationProduct # Description Pkg. Size90003 Pierce Phosphoprotein Enrichment KitIncludes: Phosphoprotein Enrichment Columns(1 ml resin bed)Lysis/Binding/Wash BufferElution BufferCHAPSProtein Concentrators (7ml/9K MWCO)White Column CapsKit10 ea.325 ml60 ml1 g10 ea.10 ea.† From 2 mg total protein.‡ Based on maximum 1 mg load per manufacturer’s protocol.70For more information, or to download product instructions, visit www.thermo.com/pierce

Pierce ConA and WGA Glycoprotein Isolation KitsIsolate glycoproteins from complex protein mixtures.Glycosylation is a post-translational modification that plays animportant role in biological functions, including immune regulation,inflammation, cell-to-cell adhesion, and cell signaling. We offertwo lectin-based glycoprotein isolation kits, concanavalin A (ConA)and wheat germ agglutinin (WGA), that allow isolation of glycoproteinsfrom complex protein mixtures, including serum, tissueand cultured cell lysates, thus enabling downstream analysis. ConAlectin recognizes α-linked mannose and terminal glucose residues,while WGA lectin selectively binds to N-Acetyl glucosamine(GlcNAc) groups and to sialic acid.Highlights:• High recovery – equivalent or greater glycoprotein recoveryvs. competitor kits and lectin resins• Fast – glycoprotein purification in less than one hour• Versatile – isolate glycoproteins from various sample types;e.g., human serum and cell lysate• Robust – lectin does not leach from resin when processing sample• Convenient – complete kit offering lectin resins and spin columnswith all necessary reagents• Compatible with Bradford-based protein assays – dialysis orprotein precipitation of recovered glycoproteins is not requiredprior to protein assayHuman SerumPierce KitSupplier ACHO LysatePierce KitSupplier AGlycoprotein isolation from human serum andcell lysate: performance comparison of kitsusing WGA resin. Human serum and CHO lysatesamples were processed with the ThermoScientific Pierce Glycoprotein Isolation Kit, WGAand with another commercially available WGAresin. An equivalent amount of total protein wasapplied to each resin. Eluted glycoprotein fractionswere normalized by volume, resolved on 8–16% polyacrylamide gels and silver stained.Human Serum CHO Lysate Glycoprotein isolation fromhuman serum and cell lysate:performance comparison ofPierce KitSupplier ASupplier GConAPierce KitSupplier ASupplier GConAkits using ConA resin. Humanserum and CHO lysate sampleswere processed with theThermo Scientific PierceGlycoprotein Isolation Kit andother commercially availableConA resins. An equivalentamount of total protein wasapplied to each resin. Elutedglycoprotein fractions werecompared with ConA Resinboiled in SDS-PAGE Buffer torelease lectins. All fractions werenormalized by volume, resolvedon 8–16% polyacrylamide gelsalongside purified ConA(rightmost lanes) then silver-stained. Arrows identify protein bands that result fromConA leaching from the resin during the elution process.Ordering InformationProduct # Description Pkg. Size89804 Pierce Glycoprotein Isolation Kit, ConASufficient reagents to isolate glycoproteins withstrong affinity for ConA from 10 samples of up to640 µl (1-1.5 mg total protein) each.Includes: ConA Lectin ResinBinding/Wash Buffer, 5X Stock SolutionElution BufferSpin Columns and Accessories89805 Pierce Glycoprotein Isolation Kit, WGASufficient reagents to isolate glycoproteins withstrong affinity for WGA from 10 samples of up to640 µl (1–1.5 mg total protein) each.Includes: WGA Lectin ResinBinding/Wash Buffer, 5X Stock SolutionElution BufferSpin Columns and AccessoriesKit1.1 ml6.5 ml5 ml1 packKit1.1 ml6.5 ml5 ml1 pack1. Pipette 200 µl ofresin slurry to spincolumn. Centrifugecolumn to removestorage buffer.2. Wash resin 3Xwith 200 µl ofBinding/WashBuffer.3. Dilute glycoproteinsample inBinding/WashBuffer (4:1).4. Add diluted glycoproteinsample to column andmix for 10 minutes.5. Centrifugeand discardflow-through.6. Wash resinwith 400 µl ofBinding/WashBuffer 4X.7. Add 200 µl ofElution Bufferand mix for5 10 minutes.8. Centrifuge.Collect eluateand repeatsteps 7 and 8.Thermo Scientific Pierce Glycoprotein Isolation Kit protocol.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.71

Additional Thermo Scientific Affinity Supports and KitsPierce Ubiquitin Enrichment KitCapture more ubiquitin-modified proteins!The Thermo Scientific Pierce Ubiquitin Enrichment Kit facilitates theisolation of polyubiquitin protein conjugates from cultured cells andtissue samples. The enriched fraction can subsequently be analyzedto determine the amount of general ubiquitin conjugates presentor to identify a specific protein of interest by Western blotting. Theassay protocol is fast and straightforward, allowing for isolationof polyubiquitinated proteins and the fractionation of monoubiquitinatedspecies in the resin flow-through. The Ubiquitin EnrichmentKit outperforms kits from other suppliers and provides a clean andspecific preparation of proteins when compared to a control resin.Highlights:• Fast – less than 45 minutes hands-on time• Complete – includes all reagents needed for ubiquitin-modifiedprotein enrichment from cultured cells and tissue samples• Flexible – sample incubation from 2 hours to overnight allowsassay to be completed in several hours or in less than 30 minutesafter overnight incubation• Robust – compatible with all Pierce Cell Lysis Products andstandard RIPA formulations• Multiple-sample format – easily processes 1–15 samples concurrentlyThermo Scientific Pierce Ubiquitin Enrichment Kit protocol.The ubiquitin proteasome pathway is the principal non-lysosomalpathway that controls the proteolysis. This pathway is significantlyCell or tissue lysate150 µg (150 µl)involved in a variety of cellular processes, including DNA repair,transcriptional regulation, signal transduction, cell metabolismand morphogenesis. Differences in total ubiquitination or theubiquitination of specific proteins affect numerous pathologicalconditions, including malignancies, certain genetic diseases andneurodegenerative diseases.kDa2201201008060504020ThermoScientificKitSupplierCAb-basedMethodGSHResinH F E F E F E F EThermo Scientific Pierce Ubiquitin Enrichment Kit recovers more ubiquitin-modifiedproteins than any other available method. Western blot imagereflects detection of samples using the anti-ubiquitin antibody supplied in thekit. Epoxomicin-treated HeLa cells lysates (150 µg) were processed by fourdifferent methods. The resulting flow-through (F) and elution (F) fractions werevolume-normalized to the original unprocessed lysate (H) and identical volumeselectophoresed for Western blot detection. Compared to Supplier C’s Kit andan antibody-based method, the Pierce Kit yielded more ubiquitinated proteinin the elution fraction (and less protein in the flow-through fraction), indicatingsignificantly better enrichment of ubquitinated proteins. GSH Resin is a negativecontrol for comparison.Ordering InformationAdd 150 µl sampledilution buffer and 20 µlubiquitin affinity resinIncubate at 4˚C for2 hours or overnightCentrifuge spin columnat 5,000 x g for 15 secondsFlow-throughResinSave for analysisProduct # Description Pkg. Size89899 Pierce Ubiquitin Enrichment KitSufficient materials for enriching up to 15 lysatesamples containing ~0.15 mg total protein per sample.Includes: Pack 1Polyubiquitin Positive Control (1,000X)Anti-ubiquitin Antibody (rabbit antiserum)Pack 2Polyubiquitin Affinity Resin, supplied asa 25% slurryBinding Capacity: ~1 µg per 20 µl of slurryTris Buffered Saline PackSpin Columns and AccessoriesKit50 µl50 µl300 µl1 each18 columnsSave washesfor analysisWash resin 3Xwith wash bufferElute ubiquitinated proteinsample with SDS-PAGEloading buffer or IEF bufferPerform Western analysis usinganti-ubiquitin antisera (included with kit)or antibody for your protein of interest72For more information, or to download product instructions, visit www.thermo.com/pierce

HisPur Cobalt Resin, Spin Columns,and Chromatography CartridgesCo 2 +Ni 2 +Co 2 +Ni 2 +Co 2 +Ni 2 +1 2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6 LSpecific, fast and gentle purification of His-tagged proteins.The preferred method for purifying recombinant His-taggedproteins is immobilized metal affinity chromatography (IMAC).Traditionally, chelating chromatography resins are charged witheither nickel or cobalt ions that coordinate with the histidine sidechains in the 6xHis-tag. HisPur Cobalt Resin is a tetradentatechelating resin charged with cobalt that binds His-tagged proteinswith high specificity and releases them under lower imidazoleconcentrations than required with nickel resins (see Figure).HisPur Cobalt Resin can be used to obtain high-purity proteinswith no metal contamination.The HisPur Chromatography Cartridges are convenient, reliableand ready-to-use pre-packed devices for the isolation of proteinsin solution and purification of His-tagged fusion proteins. TheHisPur Cartridges are compatible with automated LC instrumentation,such as the ÄKTÄ and BioLogic Systems, and adapt tomanual syringe processing.Highlights:• High purity – obtain pure protein without optimizing imidazolewashing conditions• Specificity – cobalt:chelate binding core binds fewer contaminants,resulting in lower background than nickel (see Table)• Low metal leaching – no metal contamination in eluted sample• Versatility – purify proteins under native or denaturing conditions;compatible with cell lysis reagents and a variety of buffer additives• Flexibility – available as bulk resin or predispensed columnscompatible with both spin and gravity-flow formats• Cost effective – reuse or discard• Superior – performs better than other commercially availableIMAC Resins (see Figure)WashElutionGFP β-gal Protein LThermo Scientific HisPur Cobalt Resin outperforms other IMAC resins. E. colilysates containing overexpressed His-tagged green fluorescent protein (GFP),β-galactosidase or Protein L were applied to 200 µl bed volumes of HisPurCobalt Resin and several competing cobalt and nickel resins. Thefirst elution fraction for each IMAC resin was analyzed by SDS-PAGE andprotein purity determined. Gel lanes were normalized to equivalent volume.Lane 1 = HisPur Cobalt Resin, Lane 2 = supplier C’s cobalt resin, Lane 3 =supplier S’s cobalt resin, Lane 4 = supplier G’s nickel resin, Lane 5 = supplierQ’s nickel resin, Lane 6 = Ni 2+ -IDA resin and Lane L = lysate load.Ordering InformationProduct # Description Pkg. Size89967 HisPur Cobalt Spin Columns, 0.2 ml 25 columns89968 HisPur Cobalt Spin Columns, 1 ml 5 columns89969 HisPur Cobalt Spin Columns, 3 ml 5 columns89964 HisPur Cobalt Resin 10 ml bottle89965 HisPur Cobalt Resin 100 ml bottle89966 HisPur Cobalt Resin 500 ml bottle90090 HisPur Purification Kit, 0.2 ml 25 columns90091 HisPur Purification Kit, 1 ml 5 columns90092 HisPur Purification Kit, 3 ml 5 columns90093 HisPur Chromatography Cartridges 5 x 1 ml90094 HisPur Chromatography Cartridges 2 x 5 mlkDa M F 1 2 3 1 2 3 4 5 L21011047322516.56xHis-GFPThermo Scientific HisPur Cobalt Resin is specific for His-tagged proteins andallows mild, efficient elutions. Bacterial lysate (1.0 mg total protein) was applied toa 200 µl bed volume of HisPur Cobalt Resin in a spin column. Gel lanes were normalizedto equivalent volume. M = Molecular Weight Markers (Product # 26691), L =lysate load and F = flow-through.Thermo Scientific HisPur Cobalt Resin yields more His-tagged protein and higher purity than other Co 2+ and Ni 2+ IMAC Resins.His-GFP His-β-Gal His-Protein LYield (µg)* Purity (%)** Yield (µg)* Purity (%)** Yield (µg)* Purity (%)**Thermo Scientific HisPur Cobalt Resin 298 87 78 93 42 77Supplier C Cobalt Resin 206 78 26 90 35 76Supplier S Cobalt Resin 211 85 27 65 38 77Supplier G Nickel Resin 239 84 42 83 29 68Supplier Q Nickel Resin 242 85 24 48 30 72Ni 2+ -IDA Resin 70 37 6 16 17 46* Recovered from a 5 mg total protein load (total protein yield x purity).** Purity of the first elution fraction.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.73

Additional Thermo Scientific Affinity Supports and KitsMagnaBind Beads and SupportsA convenient method for isolating biomolecules using affinitybinding, while retaining biological activity.Magnetic beads are a convenient affinity support for a varietyof assays, which allow easy purification of the target withoutcolumns or centrifugation. After a binding step in an affinitypurification procedure, the magnetic particles are easily andrapidly collected by placing the microcentrifuge tube or reactionvessel next an appropriate rare-earth magnet (see Figure).Thermo Scientific MagnaBind beads respond rapidly toMagnaBind Magnets but can be easily dispersed and regatheredmultiple times (i.e., they will not irreversibly aggregate) becausethey do not have any magnetic memory. MagnaBind Beads areavailable pre-coated with Protein A, Protein G, streptavidin, antimouseor anti-rabbit antibodies. Activated beads, with eitheramine or carboxyl groups, are also available for attaching otherproteins or affinity ligands to a magnetic particle.Highlights:• Available pre-coated with popular affinity ligands or derivatizedfor covalent attachment of proteins and other specific ligands• Beads do not irreversibly aggregate because they have nomagnetic memory; collect and disperse the beads multiple timesif needed• Most separations require a short five- to 10-minute bench-topprocedureApplications:• Cell sorting using positive or negative selection• Protein purification or immunoassays using direct or indirect methodsCharacteristics of underivatized Thermo Scientific MagnaBind Beads.CompositionMagnetizationType of MagnetizationSilanized iron oxide25–35 EMU/gSurface Area > 100 m 2 /gSettling RateEffective DensityNumber of BeadsSuperparamagnetic (no magnetic memory)4% in 30 minutes2.5 g/ml1 x 10 8 beads/mgpH Stability Aqueous solution, above pH 4.0Concentration5 mg/mlNote: To establish a microbe-free preparation, MagnaBind Beads can be washed withantibiotic medium or γ-irradiated.Thermo Scientific MagnaBind Magnet for 1.5 ml Microcentrifuge Tube. ThermoScientific MagnaBind Beads in solutions within a microcentrifuge tube are rapidly“pelleted” when the tube is placed in the magnetized holder. Magnets forsix microcentrifuge tubes and 96-well microplates are also available.ReferencesChaudhuri, T.K., et al. (2001). Cell 107, 235–246.Newey, S.E., et al. (2001). J. Biol. Chem. 276, 6645–6655.Xu, X., et al. (2001). J. Biol. Chem. 276, 43221–43230.Ordering InformationProduct # Description Pkg. Size21344 MagnaBind Streptavidin Beads 5 ml21348 MagnaBind Protein A Beads 5 ml21349 MagnaBind Protein G Beads 5 ml21354 MagnaBind Goat Anti-Mouse IgG Beads 50 ml74For more information, or to download product instructions, visit www.thermo.com/pierce

21356 MagnaBind Goat Anti-Rabbit IgG Beads 50 ml21353 MagnaBind Carboxyl Derivatized Beads 5 ml21352 MagnaBind Amine Derivatized Beads 5 ml21358 MagnaBind Magnet for 96-Well PlateSeparator21357 MagnaBind Magnet for 1.5 mlMicrocentrifuge Tube21359 MagnaBind Magnet for 6 x 1.5 mlMicrocentrifuge Tubes1 magnet1 magnet1 magnetPolystyrene Hydrazide BeadsIdeal for coupling antibodies.Highlights:• This method couples antibodies via carbohydrate groups in theFc region, resulting in site-directed immobilization in one step• Coupling of IgG to Hydrazide Beads can also be done viaglutaraldehyde activation and reduction of the Schiff base,upon introduction of an amine-containing species with NaCNBH 3ReferenceO’Shannessy, D.J. and Hofmann, W.L. (1987). Biotech. Appl. Biochem. 9, 488–496.Ordering InformationProduct # Description Pkg. Size20202 Hydrazide BeadsHydrazide-derivatized, nonporous sphericalpolystyrene beadsBead Diameter: 1/4”Loading: approximately 3 µmol of hydrazidefunction per bead250 beadsOOHOBeadNHNH 2BeadNHNSpacerArmHydrazideGroupAntibody with CarbohydrateGroups Oxidized to FormAldehyde GroupsHydrazoneBondHydrazide Bead StructureOxidized GlycoproteinImmobilized GlycoproteinTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.75

Thermo Scientific Affinity SupportsProduct Type Product Name Product # Pkg. SizeAntibody Purification – Purify a wide variety of monoclonal and polyclonal antibodies from serum, culture supernatant or ascites fluid.Protein A Protein A Agarose 20333, 20334 5 ml resin, 25 ml resinProtein A Columns 20356 5 x 1 ml columnsProtein A IgG Purification Kit 44667 5 x 1 ml columns + reagentsProtein A Trisacryl Resin 20338 5 ml resinProtein A UltraLink Resin 1 53139 5 ml resinProtein A Plus Agarose 22810, 22811, 22812 5 ml resin, 25 ml resinNab Protein A Plus Spin Columns 89952, 89956, 89960 0.2, 1, 5 ml columnsProtein A Plus UltraLink Resin 1 53142 5 ml resinRecombinant Protein A Agarose 20365, 20366 5 ml resin, 25 ml resinNAb Protein A Spin Kit 89948, 89978 resin + reagentsMagnaBind Protein A Beads 21348 5 mlProtein G Protein G Agarose 20398, 20399 2 ml resin, 10 ml resinNAb Protein G Spin Columns 89953, 89957, 89961 2, 1, 5 mlProtein G UltraLink Resin 1 53125, 53126 2 ml resin, 10 ml resinProtein G UltraLink Columns 1 53127 2 x 2 ml columnsProtein G Plus Agarose 22851, 22852 2 ml resin, 10 ml resinProtein G Plus UltraLink Resin 1 53128 2 ml resinNAb Protein G Spin Kit 89949, 89979 resin + reagentsMagnaBind Protein G Beads 21349 5 mlProtein A/G Protein A/G Agarose 20421, 20422 3 ml resin, 15 ml resinNAb Protein A/G Spin Columns 89954, 89958, 89962 2, 1, 5 mlNAb Protein A/G Spin Kit 89950, 89980 resin + reagentsProtein A/G UltraLink Resin 1 53132, 53133 2 ml resin, 10 ml resinProtein A/G Plus Agarose 20423 2 ml resinProtein A/G Plus UltraLink Resin 1 53135 2 ml resinProtein L Protein L Agarose 20510, 20512 2 ml resinNAb Protein L Spin Columns 89955, 89959, 89963 0.2, 1, 5 ml columnsNAb Protein L Spin Kit 89951, 89981 resin + reagentsProtein L Plus Agarose 20520 2 ml resinProtein L Chromatography Cartridge 89928, 89929 2 x 1 ml, 1 x 5 mlPierce Thiophilic Products Pierce Thiophilic Adsorbent 20500 10 ml resinPierce Thiophilic Purification Kit 44916 4 x 3 ml column + reagentsMelon Gel Kits Melon Gel IgG Spin Purification Kit 45206 25 spin columns + reagentsMelon Gel IgG Purification Kit 45212 25 ml resin + reagentsMelon Gel Monoclonal IgG Purification Kit 45214 200 ml resin + reagentsJacalin Immobilized Jacalin 20395 5 ml resinMBP Immobilized MBP 22212 10 ml resinIgM Purification Kit 44897 1 x 5 ml column + reagentsUltraLink Immobilized MBP 1 52123 5 ml resinProtein Isolation Kits – Isolate important subcellular components.Cell Surface Isolation Cell Surface Protein Isolation Kit 89881 8 spin columns + reagentsPhosphoprotein Enrichment Phosphoprotein Enrichment Kit 90003 10 spin columns + reagentsUbiquitin Enrichment Ubiquitin Enrichment Kit 89899 15 spin columns + reagentsGlycoprotein Isolation Glycoprotein Isolation Kit, ConA 89804 10 spin columns + reagentsGlycoprotein Isolation Kit, WGA 89805 10 spin columns + reagentsContaminant Antibody Removal – Minimize background problems by removing cross-reactive antibodies.Anti-E. coli antibodies Immobilized E. coli Lysate 44938 5 ml resinImmobilized E. coli Lysate Kit 44940 2 ml column + reagentsAnti-GST antibodies Immobilized GST 20205 2x2 ml columnImmobilized GST 20211 5 ml resinImmunoprecipitation/Pull-down – Cleanly and efficiently isolate interacting proteins using these innovative products.Immunoprecipitation Kits Seize Primary IP Kit 45335 Reagents for 20+ IPsSeize Primary Mammalian IP Kit 45332 Reagents for 20+ IPsSeize X Protein A IP Kit 45215 Reagents for 40 IPsSeize X Protein G IP Kit 45210 Reagents for 40 IPsPierce Classic Protein A IP Kit 45213 Reagents for 50 IPsPierce Classic Protein G IP Kit 45218 Reagents for 50 IPsPierce Co-IP Kit 23600 Reagents for 40 IPsPierce Mammalian Co-IP Kit 23605 Reagents for 40 IPsPull-down Kits Pierce Pull-Down PolyHis Protein:Protein Interaction Kit 21277 Reagents for 25 pull-downsPierce Pull-Down GST Protein:Protein Interaction Kit 21516 Reagents for 25 pull-downsPierce Pull-Down Biotinylated Protein:Protein Interaction Kit 21115 Reagents for 25 pull-downsMagnetic Isolation MagnaBind Goat Anti-Mouse IgG Beads 21354 50 mlMagnaBind Goat Anti-Rabbit IgG Beads 21356 50 mlMagnaBind Streptavidin Beads 21344 5 ml76For more information, or to download product instructions, visit www.thermo.com/pierce

Support Approximate Binding Capacity Applications and Features6% agarose 12–19 mg human IgG/ml resin Purify monoclonal and polyclonal IgG from serum, culture supernatant or ascites fluid6% agarose 12–19 mg human IgG/ml resin Ideal support to purify rabbit IgG6% agarose 12–19 mg human IgG/ml resinTrisacryl ® GF-2000> 15 mg human IgG/ml resinPolyacrylamide> 16 mg human IgG/ml resin6% agarose > 35 mg human IgG/ml resin6% agarose > 35 mg human IgG/ml resinPolyacrylamide> 30 mg human IgG/ml resin6% agarose > 12 mg human IgG/ml resin6% agarose ~1 mg IgG/purificationIron oxide > 0.2 mg IgG/ml resin Save time by performing immunoprecipitation magnetically6% agarose 11–15 mg human IgG/ml resin Purify monoclonal and polyclonal IgG from serum, culture supernatant or ascites fluid6% agarose 11–15 mg human IgG/ml resin Broader species specificity than Protein A with strong binding to Mouse IgG1 and human IgG3Polyacrylamide > 20 mg human IgG/ml resin Binds only IgG isotype antibodiesPolyacrylamide> 20 mg human IgG/ml resin6% agarose > 20 mg human IgG/ml resinPolyacrylamide> 25 mg human IgG/ml resin6% agarose ~1 mg IgG/purificationIron oxide > 0.2 mg IgG/ml resin Save time by performing immunoprecipitation magnetically6% agarose > 7 mg human IgG/ml resin Purify monoclonal and polyclonal IgG from serum, culture supernatant or ascites fluid6% agarose > 7 mg human IgG/ml resin6% agarose > 7 mg human IgG/ml resin Broadest specificity-combines the binding specificity of Protein A and Protein GPolyacrylamide> 20 mg human IgG/ml resin6% agarose > 50 mg human IgG/ml resinPolyacrylamide> 28 mg human IgG/ml resin6% agarose 5–10 mg human IgG/ml resin Purify monoclonal and polyclonal antibodies of all classes from serum, culture supernatant or ascites fluid6% agarose 5–10 mg human IgG/ml resin Purify single-chain variable fragments (ScFv) or Fab fragments6% agarose 5–10 mg human IgG/ml resin Binds only to antibodies with specific kappa light chains (mouse k1, human k1, k3, k4)6% agarose 10–20 mg human IgG/ml resin6% agarose 5–10 mg human IgG/ml resin6% agarose ~20 mg Ig/ml resin Purify antibodies from serum, culture supernatants or ascites fluid of a wide variety of species6% agarose ~20 mg Ig/ml resin Gentle elution conditions preserve antibody activityAgarose Purify ~1 mg IgG/spin column Purify IgG from serum in 15 minutes without loss of activityAgarosePurify ~2 g IgG/kitAgarose Purify ~1 L culture supernatant Purify IgG from culture supernatant or ascites without loss of activity6% agarose 1–3 mg human IgA/ml resin Purify human IgA without contaminating IgG or IgM4% agarose ~1 mg IgM/ml resin Purify IgM in a single step4% agarose ~1 mg IgM/ml resinPolyacrylamide> 0.75 mg IgM/ml resin6% agarose 10–40 million cells/sample Label and purify cell surface proteins from cultured mammalian cellsAgarose ~300 µg phosphoprotein/sample Isolate phosphoproteins from mammalian cells and tissuesAgarose ~0.15 mg protein/sample Isolate poly-ubiquitin modified proteins from cell or tissue lysatesAgarose ~1.5 mg mg protein/sample Isolate glycoproteins with a strong affinity for ConA (mannose and glucose)Agarose ~1.5 mg mg protein/sample Isolate glycoproteins with a strong affinity for WGA (sialic acid and GlcNAc)4% agarose ~1 mg E. coli lysate/ml resin Remove E. coli-reactive antibodies to reduce background when screening4% agarose ~1 mg E. coli lysate/ml resin6% agarose ~0.5 mg anti-GST/ml resin Prepare antibodies specific to the fusion protein rather than to the fusion tag by removing the GST-reactive antibodies6% agarose ~0.5 mg anti-GST/ml resin6% agarose 25–400 µg antibody/IP Isolate proteins using any antibody and without antibody bands interfering with protein analysis on the gel6% agarose 25–400 µg antibody/IP Enhances co-immunoprecipitation experiments by removing antibody bands from the analysis6% agarose 50–500 µg antibody/IP Isolate proteins using antibodies that bind to Protein A or G without antibody bands interfering with protein analysis on the gel6% agarose 50–500 µg antibody/IP Enhances co-immunoprecipitation experiments by removing antibody bands from the analysis6% agarose 50–500 µg antibody/IP Convenient format increases washing efficiency and requires less time than traditional immunoprecipitation6% agarose 50–500 µg antibody/IP6% agarose 25–400 µg antibody/IP Enhances co-immunoprecipitation experiments by removing antibody bands from the analysis6% agarose 25–400 µg antibody/IP Control procedures ensure that interactions are real4% agarose > 10 mg fusion protein/ml resin Isolate interacting proteins more efficiently with a tagged bait protein4% agarose > 8 mg fusion protein/ml resin Saves time compared to traditional pull-down experiments4% agarose > 5 mg biotinylated BSA/ml resinIron oxide ~0.2 mg mouse IgG/ml resin Save time by performing immunoprecipitation or pull-down experiments magneticallyIron oxide~0.2 mg rabbit IgG/ml resinIron oxide~2 µg biotin/ml resinTo order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.77

Thermo Scientific Affinity SupportsProduct Type Product Name Product # Pkg. SizeActivated Affinity Supports – Immobilize virtually any molecule on a solid support. Then use it in a batch or column method to purify its binding partners.Amine-reactive AminoLink Plus Immobilization Kit 44894 5 x 2 ml columns + reagentsAminoLink Plus Coupling Resin 20501 10 ml resinAminoLink Kit 44890 5 x 2 ml columns + reagentsAminoLink Coupling Resin 20381, 20382 10 ml resin, 50 ml resinUltraLink Biosupport 53110 2 ml resinUltraLink Biosupport 53111 10 ml resinPierce CDI Support 20259 10 ml resin, 50 ml resinPierce CDI Trisacryl (GF-2000) Support 20377 50 ml resinAminoLink Plus Micro Protein Coupling Kit 20475 10 coupling reactionsSulfhydryl-reactive SulfoLink Kit for Proteins 44995 5 x 2 ml columns + reagentsSulfoLink Kit for Peptides 44999 5 x 2 ml columns + reagentsSulfoLink Coupling Resin 20401, 20402 10 ml resin, 50 ml resinUltraLink Iodoacetyl 1 53155 10 ml resinUltraLink Iodoacetyl Micro Peptide Coupling Kit 20485 10 coupling reactionsCarbohydrate-reactive CarboLink Kit 44900 5 x 2 ml columns + reagentsCarboLink Coupling Resin 20391 10 ml resinUltraLink Hydrazide 53149 10 ml resinCarboxyl-reactive CarboxyLink Coupling Kit 44899 5 x 2 ml column + reagentsCarboxyLink Coupling Resin 20266 25 ml resinCarboxyLink Coupling Kit with UltraLink Resin 53154 5 x 2 ml column + reagentsActive hydrogen-reactive PharmaLink Immobilization Kit 44930 5 x 2 ml column + reagentsAntibody Orientation Protein A IgG Plus Orientation Kit 44893 2 x 2 ml column + reagentsProtein G IgG Plus Orientation Kit 44990 2 x 2 ml column + reagentsGST Orientation GST Orientation Kit 78201 2 x 2 ml column + reagentsFusion Protein Purification – Efficiently purify GST-, PolyHis- or MBP-tagged fusion proteins in a single step.GST-tagged Immobilized Glutathione 15160 10 ml resinB-PER GST Fusion Protein Column Purification Kit 78200 5 x 1 ml columns + reagentsB-PER GST Fusion Protein Spin Purification Kit 78400 16 spin columns + reagentsY-PER GST Fusion Protein Column Purification Kit 78997 5 x 1 ml columns + reagentsPolyHis tagged HisPur Cobalt Resin 89964, 89965, 89966 10 ml, 100 ml, 500 ml resinHisPur Cobalt Spin Columns 89967, 89968, 89969 25 x 0.2, 5 x 1, 5 x 3 ml columnsPierce Nickel Chelated Plate 2 75824 96-well plate + discsPierce Nickel Chelated Discs 2 89827 96 discsB-PER 6xHis Fusion Protein Column Purification Kit 78100 5 x 1 ml columns + reagentsB-PER 6xHis Fusion Protein Spin Purification Kit 78300 16 spin columns + reagentsY-PER 6xHis Fusion Protein Column Purification Kit 78994 5 x 1 ml columns + reagentsAvidin-Biotin – Purify or immobilize biotinylated molecules through their interaction with avidin.Avidin Avidin Agarose 20219, 20225 5 ml resin, 25 ml resinAvidin Columns 20362 5 x 1 ml columnsStreptavidin Streptavidin Agarose 20347, 20349, 20353 2 ml resin, 5 ml resin, 10 ml resinStreptavidin Columns 20351 5 x 1 ml columnsUltraLink Immobilized Streptavidin 1 53113, 53114 2 ml resin, 5 ml resinUltraLink Immobilized Streptavidin Plus 1 53116, 53117 2 ml resin, 5 ml resinMagnaBind Streptavidin Beads 21344 5 mlNeutrAvidin Biotin-Binding Protein NeutrAvidin Agarose 29200, 29201 5 ml resin, 10 ml resinUltraLink Immobilized NeutrAvidin 1 53150 5 ml resinUltraLink Immobilized NeutrAvidin Plus 1 53151 5 ml resinMonomeric Avidin Monomeric Avidin Agarose 20228 5 ml resinMonomeric Avidin Agarose Kit 20227 2 ml column + reagentsUltraLink Immobilized Monomeric Avidin 1 53146 5 ml resinBiotin Biotin Agarose 20218 5 ml resinIminobiotin Agarose 20221 5 ml resin78Specialized Affinity Supports – Purify, or remove from solution, a variety of biologically important molecules.Phosphorylated Peptides Phosphopeptide Isolation Kit 89853 30 isolationsAlbumin Pierce Blue Albumin Removal Kit 2 89845, 89846 25 discs, 100 discs + reagentsHeparin-binding Proteins Immobilized Heparin Gel 20207 10 ml resinC-reactive Protein Immobilized P-Aminophenyl Phosphoryl Choline 20307 5 ml resinLectins Immobilized D-Galactose 20372 5 ml resinGlycoproteins Immobilized Boronic Acid Gel 20244 10 ml resinHis-tagged Proteins Immobilized Iminodiacetic Acid 20277 10 ml resinProteases Immobilized Pepstatin 20215 5 ml resinImmobilized Soybean Trypsin Inhibitor 20235 2 ml resinEndotoxin DetoxiGel Prepacked Columns 20344 5 x 1 ml columnsDetoxiGel Endotoxin Removing Gel 20339, 20340 10 ml resin, 1,000 ml resinDetergent Detergent Removing Gel 20208, 20303 10 ml resin, 100 ml resinAll products are available in bulk quantities upon request.1. UltraLink Support is a copolymer of polyacrylamide and azlactone with high surface area, large pore volume and low nonspecific binding. It is suitable for pressures up to 100 psi and linear flow rates up to 3,000 cm/hour.For more information, or to download product instructions, visit www.thermo.com/pierce

Support Approximate Binding Capacity Applications and Features4% agarose Range of 1–25 mg protein/ml resin Immobilize any protein through exposed lysine residues onto a rigid support for higher flow rates4% agarose 0.1–2 mg peptide/ml resin Stable, uncharged linkage for maximum binding specificity4% agarose Range of 1–20 mg protein/ml resin Immobilize any protein through exposed lysine residues4% agarose 0.1–2 mg peptide/ml resin Stable, uncharged linkage for maximum binding specificityPolyacrylamide Range of 1–30 mg protein/ml resin Immobilize any protein through exposed lysine residuesPolyacrylamide 0.1–2 mg peptide/ml resin Rigid support for medium pressure applications6% agarose Range of 1–10 mg protein/ml resin Immobilize any protein through exposed lysine residuesTrisacryl ® GF-2000 0.1–2 mg peptide/ml resin Stable, uncharged linkage for maximum binding specificity4% agarose ~100 µg protein6% agarose Range of 1–10 mg protein/ml resin Immobilize peptides with a terminal cysteine residue for antibody purification6% agarose6% agarose 0.1–2 mg peptide/ml resin Long spacer arm reduces steric hindrance for maximal antibody bindingPolyacrylamideImmobilize proteins that contain free cysteine residuesPolyacrylamide~250 µg peptide6% agarose Range of 1–5 mg glycoprotein/ml resin Immobilize polyclonal antibodies and other glycoproteins6% agarose Antibodies are oriented properly for maximum binding because attachment is through the Fc regionPolyacrylamide4% agarose Range of 1–10 mg protein/ml resin Immobilize peptides/proteins through aspartic and glutamic acid residues or the carboxy terminus4% agarose 0.1–2 mg peptide/ml resin Long spacer arm reduces steric hindrancePolyacrylamide6% agarose Variable, up to 20 µmol/ml gel Immobilize drugs and other organic molecules with no available reactive groups6% agarose Up to 16 mg rabbit IgG/column Purify and covalently immobilize an antibody with a single column6% agarose Up to 16 mg rabbit IgG/column4% agarose 1–10 mg fusin protein/column Purify and covalently immobilize GST fusion proteins with a single column4% agarose ~10 mg fusion protein/ml resin Purify GST-tagged fusion proteins4% agarose ~ 10 mg fusin protein/column Purify GST-tagged fusion proteins4% agarose ~1 mg fusion protein/spin column Kit includes lysis reagent for optimal protein recovery4% agarose ~10 mg fusion protein/column6% agarose ~ 10 mg fusion protein/ml resin Purify His-tagged fusion proteins6% agarose ~ 10 mg fusion protein/ml resinAgarose~1 mg 6xHis-GFP/wellAgarose> 2 mg 6xHis-GFP/disc4% agarose ~10 mg fusion protein/column Purify His-tagged fusion proteins4% agarose ~1 mg fusion protein/spin column Kit includes lysis reagent for optimal protein recovery4% agarose ~10 mg fusion protein/column6% agarose > 20 µg biotin/ml resin Isolate or immobilize biotinylated proteins, peptides, nucleic acids and other molecules6% agarose > 20 µg biotin/ml resin6% agarose 1–3 mg biotinylated BSA/ml resin Isolate or immobilize biotinylated proteins, peptides, nucleic acids and other molecules6% agarose 1–3 mg biotinylated BSA/column Gives lower background than avidin because it contains no carbohydratePolyacrylamide> 2 mg biotinylated BSA/ml resinPolyacrylamide> 4 mg biotinylated BSA/ml resinIron oxide ~2 µg biotin/ml resin Magnetically isolate or immobilize biotinylated proteins, peptides, nucleic acids and other molecules6% agarose > 20 µg biotin/ml resin Isolate or immobilize biotinylated proteins, peptides, nucleic acids and other moleculesPolyacrylamide 12–20 µg biotin/ml resin Gives lowest background because carbohydrate has been removed and does not contain RYD sequencePolyacrylamide> 30 µg biotin/ml resin4% agarose > 1.2 mg biotinylated BSA/ml resin Isolate or immobilize biotinylated proteins, peptides, nucleic acids and other molecules4% agarose > 1.2 mg biotinylated BSA/ml resin Reversible binding allows mild elution of biotinylated moleculesPolyacrylamide> 1.2 mg biotinylated BSA/ml resin6% agarose 2 mg avidin/ml resin Isolate or immobilize avidin molecules or conjugates6% agarose 1 mg avidin/ml resin Reversibly isolate avidin conjugates with mild elution conditionsAgarose 150 µg phosphopeptide/isolation Enrich phosphorylated peptides within a peptide digest for mass spectral analysisAgarose ~2 mg human serum albumin/disc Remove albumin from antibodies and other samples4% agarose > 0.2 mg heparin/ml resin Purify a wide variety of proteins that have affinity for heparin6% agarose > 3 mg human C-reactive protein/ml resin Purify C-reactive protein6% agarose > 8 mg castor bean lectin/ml resin Purify lectins specific for D-galactosePolyacrylamide 100 µmol boronate/ml resin Purify glycoproteins, ribonucleosides and other sugar-containing molecules4% agarose > 14 µmol metal ions/ml resin Purify His-tagged and other metal-binding proteins6% agarose 1–2 mg pepsin/ml resin Purify pepsin and cathepsins or remove them from a sample4% agarose Up to 6 mg trypsin/ml resin Purify trypsin, chymotrypsin and elastase or remove them from a sample6% agarose 2 mg endotoxin Remove endotoxin from protein and nucleic acid samples6% agarose 2 mg endotoxin/ml resinProprietary Varies among detergents – see page 56 Remove detergent from protein and nucleic acid samples2. SwellGel Support is a convenient, room temperature-stable, dehydrated agarose resin that is rapidly rehydrated when a sample is added. In a 96-well filterplate, SwellGel Resin is ideal for high-throughput purifications.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.79

AppendicesFREE Avidin-Biotin Product GuideThermo Scient fic Pierce ®Avidin Biotin Product GuideThis reference guide brings togethereverything needed to biotinylate cellsurfaceproteins, purify a biotinylatedtarget, detect a biotinylated antibodyand perform many other applications. Itincludes dozens of references along withprotocols, troubleshooting tips, selectionguides and a complete listing of availabletools. Because the Avidin-Biotin systemcan be used in so many ways, you’ll wantto keep this booklet close at hand! Torequest a free copy, log on to www.thermo.com/pierce or call800-874-3723 or 815-968-0747. Outside the United States, contactyour local branch office or distributor.Antibody Technical HandbookThermo Scient fic Pierce ®Antibody Technical HandbookThis 69-page handbook is an essentialresource for any laboratory working withantibodies. The handbook provides andoverview of antibody structure and types,as well as technical information on theprocedures, reagents and tools used toproduce, purify, fragment and labelantibodies. To request a free copy, logon to www.thermo.com/pierce or call800-874-3723 or 815-968-0747. Outside theUnited States, contact your local branchoffice or distributor.80For more information, or to download product instructions, visit www.thermo.com/pierce

Thermo Scientific B-PER Technology is protected by U.S. patent #6,174,704.Thermo Scientific PharmaLink Immobilization Kit Technology is protected by U.S. Patent #5,142,027.Thermo Scientific Pierce Blue Albumin Removal Technology is protected by U.S. Patent #6,709,743.Thermo Scientific Pierce Thiophilic Adsorbent Technology is protected by U.S. Patent #4,696,980.U.S. patent pending on Thermo Scientific Imperial Protein Stain Technology.U.S. patent pending on Thermo Scientific Melon Gel IgG Spin Purification Kit.Trisacryl ® is a trademark of Pall Corporation.Luer-Lok is a trademark of Becton, Dickinson and Company.Brij ® is a registered trademark of ICI Americas.Cibacron ® is a registered trademark of Ciba Specialty Chemicals, Inc.Triton ® is a registered trademark of Rohm & Haas Company.Tween ® is a trademark of ICI Americas.To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor.81

Contact InformationBelgium and Europe,the Middle Eastand Africa DistributorsTel: +32 53 85 71 84FranceTel: 0 800 50 82 15The NetherlandsTel: 076 50 31 880GermanyTel: 0228 9125650United KingdomTel: 0800 252 185SwitzerlandTel: 0800 56 31 40Email: perbio.euromarketing@thermofisher.comwww.thermo.com/perbioUnited StatesTel: 815-968-0747 or 800-874-3723Customer Assistance E-mail:Pierce.CS@thermofisher.comwww.thermo.com1601617 07/08© 2008 Thermo Fisher Scientific Inc. All rights reserved.These products are supplied for laboratory or manufacturingapplications only. Unless indicated otherwise on the insideback cover, all trademarks are property of Thermo FisherScientific Inc. and its subsidiaries.