Affinity Chromatography - Department of Molecular and Cellular ...

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Chapter 5 Designing affinity media using pre-activated matrices............................................ 100 Choosing the matrix ............................................................................................................................... 100 Choosing the ligand and spacer arm ......................................................................................................... 100 Choosing the coupling method ................................................................................................................. 100 Coupling the ligand ................................................................................................................................ 102 Binding capacity, ligand density and coupling efficiency ............................................................................ 103 Binding and elution conditions ................................................................................................................ 104 Coupling through the primary amine of a ligand .......................................................... 105 HiTrap NHS-activated HP, NHS-activated Sepharose 4 Fast Flow ................................................................ 105 CNBr-activated Sepharose ....................................................................................................................... 108 Immunoaffinity chromatography .............................................................................................................. 112 Coupling small ligands through amino or carboxyl groups via a spacer arm ...................................................................................................... 113 EAH Sepharose 4B and ECH Sepharose 4B .............................................................................................. 113 Coupling through hydroxy, amino or thiol groups via a 12-carbon spacer arm ...................................................................................... 116 Epoxy-activated Sepharose 6B ................................................................................................................. 116 Coupling through a thiol group .................................................................................. 120 Thiopropyl Sepharose 6B ........................................................................................................................ 120 Coupling other functional groups ............................................................................... 121 Chapter 6 Affinity chromatography and CIPP ........................................................................... 123 Applying CIPP .......................................................................................................... 124 Selection and combination of purification techniques.................................................. 124 Appendix 1 .......................................................................................................... 129 Sample preparation ................................................................................................. 129 Sample stability ..................................................................................................................................... 129 Sample clarification ............................................................................................................................... 130 Specific sample preparation steps ............................................................................. 131 Resolubilization of protein precipitates ..................................................................................................... 133 Buffer exchange and desalting .................................................................................. 134 Removal of lipoproteins ............................................................................................ 137 Removal of phenol red ............................................................................................. 137 Removal of low molecular weight contaminants .......................................................... 137 Appendix 2 .......................................................................................................... 139 Selection of purification equipment ........................................................................... 139 Appendix 3 .......................................................................................................... 140 Column packing and preparation ............................................................................... 140
Appendix 4 .......................................................................................................... 142 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa ............................................................................. 142 Appendix 5 .......................................................................................................... 143 Conversion data: proteins, column pressures .............................................................. 143 Column pressures .................................................................................................................................. 143 Appendix 6 .......................................................................................................... 144 Table of amino acids ................................................................................................ 144 Appendix 7 .......................................................................................................... 146 Kinetics in affinity chromatography ........................................................................... 146 Appendix 8 .......................................................................................................... 151 Analytical assays during purification .......................................................................... 151 Appendix 9 .......................................................................................................... 153 Storage of biological samples .................................................................................... 153 Additional reading and reference material ............................................................. 154 Ordering information ............................................................................................ 155
Page 1 and 2: Affinity Chromatography Principles
Page 3 and 4: Affinity Chromatography Principles
Page 5: Serine proteases and zymogens with
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
Page 65 and 66:
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
Page 73 and 74:
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
Page 79 and 80:
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
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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
Page 105 and 106:
Table 9 summarizes recommended liga
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Coupling through the primary amine
Page 109 and 110:
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
Page 119 and 120:
Alternative coupling solutions: Dis
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Media characteristics Product Compo
Page 123 and 124:
Ligands containing amino groups can
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:
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
Page 141 and 142:
Appendix 2 Selection of purificatio
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4. Estimate the amount of slurry (r
Page 145 and 146:
Appendix 5 Conversion data: protein
Page 147 and 148:
Middle unit residue (-H 2 0) Charge
Page 149 and 150:
Expected results when changing cond
Page 151 and 152:
Expected results with competitive e
Page 153 and 154:
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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