Affinity Chromatography Handbook

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2 Contents Introduction ............................................................................................................. 7 Symbols and abbreviations ........................................................................................................................ 8 Chapter 1 <strong>Affinity</strong> 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 <strong>Affinity</strong> 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, MabSelect ............................................................................................. 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, GSTPrep FF 16/10, Glutathione Sepharose 4 Fast Flow, Glutathione Sepharose 4B .................................................................... 42 Poly (His) fusion proteins ........................................................................................... 47 His MicroSpin Purification Module, HisTrap Kit, HiTrap Chelating HP, Chelating Sepharose Fast Flow .................................................................................................................. 47 Protein A fusion proteins ............................................................................................ 52 IgG Sepharose 6 Fast Flow ....................................................................................................................... 52 Purification or removal of serine proteases, e.g. thrombin and trypsin, and zymogens ....... 54 HiTrap Benzamidine FF (high sub), Benzamidine Sepharose 4 Fast Flow (high sub) ....................................... 54 Serine proteases and zymogens with an affinity for arginine ........................................... 58 Arginine Sepharose 4B ............................................................................................................................. 58
DNA binding proteins ................................................................................................. 60 HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow .............................................. 60 Coagulation factors .................................................................................................... 65 HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow .............................................. 65 Biotin and biotinylated substances .............................................................................. 66 HiTrap Streptavidin HP, Streptavidin Sepharose High Performance ............................................................... 66 Purification or removal of fibronectin ........................................................................... 69 Gelatin Sepharose 4B .............................................................................................................................. 69 Purification or removal of albumin ............................................................................... 70 HiTrap Blue HP, Blue Sepharose 6 Fast Flow .............................................................................................. 70 NAD + -dependent dehydrogenases and ATP-dependent kinases ....................................... 73 5' AMP Sepharose 4B, HiTrap Blue HP, Blue Sepharose 6 Fast Flow ............................................................. 73 5' AMP Sepharose 4B .............................................................................................................................. 74 HiTrap Blue HP, Blue Sepharose 6 Fast Flow .............................................................................................. 75 NADP + -dependent dehydrogenases and other enzymes with affinity for NADP + ............................. 75 2'5' ADP Sepharose 4B, Red Sepharose CL-6B ........................................................................................... 75 2'5' ADP Sepharose 4B ............................................................................................................................ 77 Red Sepharose CL-6B .............................................................................................................................. 78 Glycoproteins or polysaccharides ................................................................................. 80 Con A Sepharose 4B, Lentil Lectin Sepharose 4B, Agarose Wheat Germ Lectin .............................................. 80 Con A for binding of branched mannoses, carbohydrates with terminal mannose or glucose (aMan > aGlc > GlcNAc) .......................................................................................................... 80 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 .................................... 83 Wheat germ lectin for binding of chitobiose core of N-linked oligosaccharides, [GlcNAc(b1,4GlcNAc) > b GlcNAc] ......................................................................................................... 84 1-2 Calmodulin binding proteins: ATPases, adenylate cyclases, protein kinases, ........................ phosphodiesterases, neurotransmitters ........................................................................ 86 Calmodulin Sepharose 4B ........................................................................................................................ 86 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) ...................................................... 88 HiTrap Chelating HP, Chelating Sepharose Fast Flow, His MicroSpin Purification Module, HisTrap Kit .............. 88 Thiol-containing substances (purification by covalent chromatography) ........................... 92 Activated Thiol Sepharose 4B, Thiopropyl Sepharose 6B ............................................................................. 92 Chapter 4 Components of an affinity medium .......................................................................... 97 The matrix .............................................................................................................................................. 97 The ligand ............................................................................................................................................... 98 Spacer arms ............................................................................................................................................ 99 Ligand coupling ..................................................................................................................................... 100 Ligand specificity .................................................................................................................................. 100 3
Page 1 and 2: 18-1022-29 Edition AD Affinity Chro
Page 3: Affinity Chromatography Principles
Page 7: Appendix 4 ........................
Page 10 and 11: 8 This handbook describes the role
Page 12 and 13: 10 Affinity chromatography is also
Page 14 and 15: 12 BioProcess Media for large-scale
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: A 280 nm 0.6 0.4 0.2 0 Fig. 8. Scou
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 0.0 0 200
Page 39 and 40: Desalt and/or transfer purified IgG
Page 41 and 42: Performing a separation Column: HiT
Page 43 and 44: Fig. 21. SDS-PAGE of non-reduced sa
Page 45 and 46: Purification options Binding capaci
Page 47 and 48: Performing a separation Binding buf
Page 49 and 50: Poly (His) fusion proteins His Micr
Page 51 and 52: One step, on-column, refolding and
Page 53 and 54: Purification using HisTrap Kit HisT
Page 55 and 56:
1. Pack the column (see Appendix 3)
Page 57 and 58:
Purification options Binding capaci
Page 59 and 60:
1. Equilibrate the column with 5 co
Page 61 and 62:
Specifically bound biomolecules can
Page 63 and 64:
Page 65 and 66:
Sample: 2000 ml partially purified
Page 67 and 68:
Coagulation factors HiTrap Heparin
Page 69 and 70:
Page 71 and 72:
Purification or removal of fibronec
Page 73 and 74:
Purification examples Figure 42 sho
Page 75 and 76:
Media characteristics Ligand and de
Page 77 and 78:
Media characteristics Ligand densit
Page 79 and 80:
Page 81 and 82:
Elution buffers: use low concentrat
Page 83 and 84:
Page 85 and 86:
Lentil lectin for binding of branch
Page 87 and 88:
Page 89 and 90:
Remove proteases as quickly as poss
Page 91 and 92:
Purification example A 280 nm 1.0 0
Page 93 and 94:
Samples: 2.5 ml cell extract contai
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In Activated Thiol-Sepharose 4B the
Page 97 and 98:
Reactivation Pass one to two column
Page 99 and 100:
Chapter 4 Components of an affinity
Page 101 and 102:
If several functional groups are av
Page 103 and 104:
Chapter 5 Designing affinity media
Page 105 and 106:
Coupling the ligand 1. Prepare the
Page 107 and 108:
Increase the ligand concentration t
Page 109 and 110:
Options Product Spacer arm Coupling
Page 111 and 112:
Media characteristics Product Ligan
Page 113 and 114:
Preparation of the matrix should be
Page 115 and 116:
Immunoaffinity chromatography Immun
Page 117 and 118:
Preparation of coupling reagent Use
Page 119 and 120:
Coupling through hydroxy, amino or
Page 121 and 122:
Coupled glycylleucine % Coupled lig
Page 123 and 124:
Coupling through a thiol group Thio
Page 125 and 126:
Chapter 6 Affinity chromatography a
Page 127 and 128:
Resolution is achieved by the selec
Page 129 and 130:
For any capture step, select the te
Page 131 and 132:
Sample clarification Centrifugation
Page 133 and 134:
Examples of precipitation agents ar
Page 135 and 136:
Resolubilization of protein precipi
Page 137 and 138:
Alternative 1: Manual desalting wit
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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 20) Charge
Page 147 and 148:
Expected results when changing cond
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Expected results with competitive e
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Appendix 8 Analytical assays during
Page 153 and 154:
Appendix 9 Storage of biological sa
Page 155 and 156:
Additional reading Code No. Purific
Page 157 and 158:
Product Quantity Code No. Blue Seph
Page 159 and 160:
STREAMLINE, Sepharose, HiTrap, ÄKT
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Affinity Chromatography Handbook

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