Affinity Chromatography - Department of Molecular and Cellular ...

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Affinity chromatography is also used to remove specific contaminants, for example Benzamidine Sepharose 6 Fast Flow can remove serine proteases, such as thrombin and Factor Xa. Figure 2 shows the key stages in an affinity purification. 1. Affinity medium is equilibrated in binding buffer. 2. Sample is applied under conditions that favour specific binding of the target molecule(s) to a complementary binding substance (the ligand). Target substances bind specifically, but reversibly, to the ligand and unbound material washes through the column. 3. Target protein is recovered by changing conditions to favour elution of the bound molecules. Elution is performed specifically, using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity.Target protein is collected in a purified, concentrated form. 4. Affinity medium is re-equilibrated with binding buffer. equilibration adsorption of sample and elution of unbound material wash away unbound material elute bound protein(s) re-equilibration Absorbance begin sample application change to elution buffer >1 1-2 cv x cv 1-2 cv cv 1-2 cv Column Volumes (cv) Fig. 2. Typical affinity purification. 10
The high selectivity of affinity chromatography enables many separations to be achieved in one simple step, including, for example, common operations such as the purification of monoclonal antibodies or fusion proteins. A wide variety of prepacked columns, ready to use media, and pre-activated media for ligand coupling through different functional groups, makes affinity chromatography readily available for a broad range of applications. To save time, the HiTrap column range (Table 1) is excellent for routine laboratory scale applications in which the risk of cross-contamination between samples must be eliminated, for purification from crude samples or for fast method development before scaling up purification. HiTrap columns can be operated with a syringe, a peristaltic pump or any ÄKTAdesign chromatography system. Several HiTrap columns can be connected in series to increase purification capacity and all columns are supplied with detailed protocols for use. Table 1. HiTrap and HiPrep affinity columns for laboratory scale purification. Application Isolation of human immunoglobulins IgG, fragments and subclasses IgG, fragments and subclasses IgG, fragments and subclasses including human IgG 3 strong affinity for monoclonal mouse IgG 1 and rat IgG Avian IgY from egg yolk Mouse and human IgM Purification of fusion proteins (His) 6 fusion proteins GST fusion proteins Other Group Specific Media Albumin and nucleotide-requiring enzymes Proteins and peptides with exposed His, Cys or Trp Biotinylated substances DNA binding proteins and coagulation factors Trypsin-like serine proteases including Factor Xa, thrombin and trypsin Matrix for preparation of affinity media. Coupling via primary amines HiTrap and HiPrep columns HiTrap rProtein A FF, 1 ml and 5 ml HiTrap Protein A HP, 1 ml and 5 ml HiTrap Protein G HP, 1 ml and 5 ml MAbTrap Kit HiTrap IgY Purification HP, 5 ml HiTrap IgM Purification HP, 1 ml HisTrap Kit HiTrap Chelating HP, 1 ml and 5 ml GSTrap FF, 1 ml and 5 ml HiTrap Blue HP, 1 ml and 5 ml HiTrap Chelating HP, 1 ml and 5 ml HiTrap Streptavidin HP, 1 ml HiTrap Heparin HP, 1 ml and 5 ml HiPrep 16/10 Heparin FF, 20 ml HiTrap Benzamidine FF (high sub), 1 ml and 5 ml HiTrap NHS-activated HP, 1 ml and 5 ml 11
Page 1 and 2: Affinity Chromatography Principles
Page 3 and 4: Affinity Chromatography Principles
Page 5 and 6: Serine proteases and zymogens with
Page 7: Appendix 4 ........................
Page 10 and 11: This handbook describes the role of
Page 14 and 15: BioProcess Media for large-scale pr
Page 17 and 18: Chapter 2 Affinity chromatography i
Page 19 and 20: Avoid using magnetic stirrers as th
Page 21 and 22: pH elution A change in pH alters th
Page 23 and 24: 0.6 0.4 0.2 0 A280 pH selected for
Page 25 and 26: Situation Cause Remedy Protein does
Page 27 and 28: Chapter 3 Purification of specific
Page 29 and 30: Table 2. Relative binding strengths
Page 31 and 32: Purification examples Figure 11 sho
Page 33 and 34: MAbTrap Kit Fig. 13. MAbTrap Kit, r
Page 35 and 36: Media characteristics Ligand Compos
Page 37 and 38: A 280 nm 2.0 1.5 1.0 0.5 Column: rP
Page 39 and 40: Desalt and/or transfer purified IgG
Page 41 and 42: Performing a separation Column: Rec
Page 43 and 44: M r 97 000 66 000 45 000 30 000 20
Page 45 and 46: in The Recombinant Protein Handbook
Page 47 and 48: Cleaning These procedures are appli
Page 49 and 50: Purification examples Figures 24 an
Page 51 and 52: Nickel solution: 0.1 M NiSO 4 Bindi
Page 53 and 54: Protein A fusion proteins IgG Sepha
Page 55 and 56: Purification or removal of serine p
Page 57 and 58: Sample: 2 ml clarified E. coli homo
Page 59 and 60: Serine proteases and zymogens with
Page 61 and 62: DNA binding proteins HiTrap Heparin
Page 63 and 64:
Sample: Column: Binding buffer: Elu
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Media characteristics Ligand densit
Page 67 and 68:
Sample: Pooled and frozen human pla
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The harsh conditions required to br
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Purification or removal of albumin
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IFN-act. A units 280 nm 250 0.12 20
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Purification options Binding capaci
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Sepharose NH (CH 2) n NH N N O N N
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If detergent or denaturing agents h
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Glycoproteins or polysaccharides Co
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For complex samples containing glyc
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For complex samples containing glyc
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Chemical stability Avoid exposure t
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Proteins and peptides with exposed
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Several methods can be used to dete
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Media characteristics Composition M
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Performing a separation Binding buf
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Do not store the suspension for lon
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Sepharose has been modified and dev
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Ligand coupling Several methods are
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Couple a ligand through the least c
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Table 9 summarizes recommended liga
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Coupling through the primary amine
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Ligand and column preparation 1. Di
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Options Product Spacer Coupling con
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100 80 Coupled substance, % 60 40 2
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Coupling small ligands through amin
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Ligand coupling 1. Add the ligand s
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Alternative coupling solutions: Dis
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Media characteristics Product Compo
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Ligands containing amino groups can
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Chapter 6 Affinity chromatography a
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Resolution is achieved by the selec
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For any capture step, select the te
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Appendix 1 Sample preparation Sampl
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Remove salts from proteins with mol
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1. Filter (0.45 µm) or centrifuge
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For laboratory scale operations, Ta
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Removal of lipoproteins Lipoprotein
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Appendix 2 Selection of purificatio
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4. Estimate the amount of slurry (r
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Appendix 5 Conversion data: protein
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Middle unit residue (-H 2 0) Charge
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Expected results when changing cond
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Expected results with competitive e
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Appendix 8 Analytical assays during
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Appendix 9 Storage of biological sa
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Ordering information Product Quanti
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STREAMLINE, Sepharose, HiTrap, ÄKT
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