Affinity Chromatography - Department of Molecular and Cellular ...

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Content Introduction ............................................................................................................. 7 Symbols and abbreviations ......................................................................................................................... 8 Chapter 1 Affinity chromatography in brief ................................................................................ 9 BioProcess Media for large-scale production ................................................................. 12 Custom Designed Media and Columns ......................................................................... 12 Common terms in affinity chromatography ................................................................... 13 Chapter 2 Affinity chromatography in practice ......................................................................... 15 Purification steps ..................................................................................................................................... 15 Media selection ....................................................................................................................................... 16 Preparation of media and buffers ............................................................................................................... 16 Sample preparation and application ........................................................................................................... 17 Elution ................................................................................................................................................... 18 Flow rates ............................................................................................................................................... 21 Analysis of results and further steps ........................................................................................................... 21 Equipment selection ................................................................................................................................ 21 Troubleshooting ....................................................................................................................................... 22 Chapter 3 Purification of specific groups of molecules ............................................................ 25 Immunoglobulins ....................................................................................................... 25 IgG, IgG fragments and subclasses .............................................................................. 26 HiTrap Protein G HP, Protein G Sepharose 4 Fast Flow, MAbTrap Kit ............................................................. 28 HiTrap Protein A HP, Protein A Sepharose 4 Fast Flow, HiTrap rProtein A FF, rProtein A Sepharose 4 Fast Flow .............................................................................................................. 33 Monoclonal IgM from hybridoma cell culture ................................................................ 38 HiTrap IgM Purification HP ....................................................................................................................... 38 Avian IgY from egg yolk .............................................................................................. 40 HiTrap IgY Purification HP ........................................................................................................................ 40 Recombinant fusion proteins ...................................................................................... 42 GST fusion proteins ................................................................................................... 42 GST MicroSpin Purification Module, GSTrap FF, Glutathione Sepharose 4 Fast Flow, Glutathione Sepharose 4B ........................................................................................................................ 42 Poly (His) fusion proteins ........................................................................................... 46 His MicroSpin Purification Module, HisTrap Kit, HiTrap Chelating HP, Chelating Sepharose Fast Flow .................................................................................................................. 46 Protein A fusion proteins ............................................................................................ 51 IgG Sepharose 6 Fast Flow ........................................................................................................................ 51 Purification or removal of serine proteases, e.g. thrombin and trypsin, and zymogens ..................................................................... 53 HiTrap Benzamidine FF (high sub), Benzamidine Sepharose 4 Fast Flow (high sub) ........................................ 53 2
Serine proteases and zymogens with an affinity for arginine ........................................... 57 Arginine Sepharose 4B ............................................................................................................................. 57 DNA binding proteins................................................................................................. 59 HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow ............................................... 59 Coagulation factors .................................................................................................... 64 HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow ............................................... 64 Biotin and biotinylated substances .............................................................................. 65 HiTrap Streptavidin HP, Streptavidin Sepharose High Performance ................................................................ 65 Purification or removal of fibronectin ........................................................................... 68 Gelatin Sepharose 4B ............................................................................................................................... 68 Purification or removal of albumin ............................................................................... 69 HiTrap Blue HP, Blue Sepharose 6 Fast Flow .............................................................................................. 69 NAD + -dependent dehydrogenases and ATP-dependent kinases ....................................... 72 5' AMP Sepharose 4B, HiTrap Blue HP, Blue Sepharose 6 Fast Flow ............................................................. 72 NADP + -dependent dehydrogenases and other enzymes with affinity for NADP + ............... 74 2'5' ADP Sepharose 4B, Red Sepharose CL-6B ........................................................................................... 74 Glycoproteins or polysaccharides ................................................................................. 79 Con A Sepharose 4B, Lentil Lectin Sepharose 4B, Agarose Wheat Germ Lectin ............................................... 79 Con A for binding of branched mannoses, carbohydrates with terminal mannose or glucose (aMan > aGlc > GlcNAc) .......................................................................................................... 79 Lentil lectin for binding of branched mannoses with fucose linked a(1,6) to the N-acetyl-glucosamine, (aMan > aGlc > GlcNAc) N-acetylglucosamine binding lectins ......................................................................................................................................... 82 Wheat germ lectin for binding of chitobiose core of N-linked oligosaccharides, [GlcNAc(b1,4GlcNAc) 1-2 > b GlcNAc] ......................................................................................................... 83 Calmodulin binding proteins: ATPases, adenylate cyclases, protein kinases, phosphodiesterases, neurotransmitters ................................................. 85 Calmodulin Sepharose 4B ......................................................................................................................... 85 Proteins and peptides with exposed amino acids: His, Cys, Trp, and/or with affinity for metal ions (also known as IMAC, immobilized metal chelate affinity chromatography) ...................................................... 87 HiTrap Chelating HP, Chelating Sepharose Fast Flow, His MicroSpin Purification Module, HisTrap Kit ........................................................................................... 87 Thiol-containing substances (purification by covalent chromatography) ........................... 91 Activated Thiol Sepharose 4B, Thiopropyl Sepharose 6B .............................................................................. 91 Chapter 4 Components of an affinity medium........................................................................... 96 The matrix ............................................................................................................................................... 96 The ligand ............................................................................................................................................... 97 Spacer arms ............................................................................................................................................ 98 Ligand coupling ....................................................................................................................................... 99 Ligand specificity ..................................................................................................................................... 99 3
Page 1 and 2: Affinity Chromatography Principles
Page 3: Affinity Chromatography Principles
Page 7: Appendix 4 ........................
Page 10 and 11: This handbook describes the role of
Page 12 and 13: Affinity chromatography is also use
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
Page 71 and 72:
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
Page 81 and 82:
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
Page 89 and 90:
Proteins and peptides with exposed
Page 91 and 92:
Several methods can be used to dete
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Media characteristics Composition M
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Performing a separation Binding buf
Page 97 and 98:
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
Page 105 and 106:
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
Page 125 and 126:
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
Page 131 and 132:
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
Page 155 and 156:
Appendix 9 Storage of biological sa
Page 157 and 158:
Ordering information Product Quanti
Page 159 and 160:
STREAMLINE, Sepharose, HiTrap, ÄKT
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