Affinity Chromatography Handbook

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4 Chapter 5 Designing affinity media using pre-activated matrices ........................................... 101 Choosing the matrix ............................................................................................................................... 101 Choosing the ligand and spacer arm ........................................................................................................ 101 Choosing the coupling method ................................................................................................................ 101 Coupling the ligand ................................................................................................................................ 103 Binding capacity, ligand density and coupling efficiency ............................................................................ 104 Binding and elution conditions ................................................................................................................ 105 Coupling through the primary amine of a ligand .......................................................... 106 HiTrap NHS-activated HP, NHS-activated Sepharose 4 Fast Flow ................................................................ 106 CNBr-activated Sepharose ...................................................................................................................... 109 Immunoaffinity chromatography .............................................................................................................. 113 Coupling small ligands through amino or carboxyl groups via a spacer arm .................... 114 EAH Sepharose 4B and ECH Sepharose 4B .............................................................................................. 114 Coupling through hydroxy, amino or thiol groups via a 12-carbon spacer arm ................. 117 Epoxy-activated Sepharose 6B ................................................................................................................ 117 Coupling through a thiol group .................................................................................. 121 Thiopropyl Sepharose 6B ........................................................................................................................ 121 Coupling other functional groups ............................................................................... 122 Chapter 6 <strong>Affinity</strong> chromatography and CIPP .......................................................................... 123 Applying CIPP .......................................................................................................... 124 Selection and combination of purification techniques ................................................. 124 Appendix 1 .......................................................................................................... 128 Sample preparation ................................................................................................. 128 Sample stability ..................................................................................................................................... 128 Sample clarification ............................................................................................................................... 129 Specific sample preparation steps ............................................................................. 130 Resolubilization of protein precipitates .................................................................................................... 133 Buffer exchange and desalting .................................................................................. 133 Removal of lipoproteins ............................................................................................ 136 Removal of phenol red ............................................................................................. 136 Removal of low molecular weight contaminants .......................................................... 136 Appendix 2 .......................................................................................................... 137 Selection of purification equipment ........................................................................... 137 Appendix 3 .......................................................................................................... 138 Column packing and preparation ............................................................................... 138
Appendix 4 .......................................................................................................... 140 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa ............................................................................. 140 Appendix 5 .......................................................................................................... 141 Conversion data: proteins, column pressures .............................................................. 141 Column pressures .................................................................................................................................. 141 Appendix 6 .......................................................................................................... 142 Table of amino acids ................................................................................................ 142 Appendix 7 .......................................................................................................... 144 Kinetics in affinity chromatography ........................................................................... 144 Appendix 8 .......................................................................................................... 149 Analytical assays during purification .......................................................................... 149 Appendix 9 .......................................................................................................... 151 Storage of biological samples ................................................................................... 151 Product index ...................................................................................................... 152 Additional reading ............................................................................................... 153 References .......................................................................................................... 153 Ordering information ............................................................................................ 154 5
Page 1 and 2: 18-1022-29 Edition AD Affinity Chro
Page 3 and 4: Affinity Chromatography Principles
Page 5: DNA binding proteins ..............
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
Page 95 and 96:
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
Page 139 and 140:
Appendix 2 Selection of purificatio
Page 141 and 142:
4. Estimate the amount of slurry (r
Page 143 and 144:
Appendix 5 Conversion data: protein
Page 145 and 146:
Middle unit residue (-H 20) Charge
Page 147 and 148:
Expected results when changing cond
Page 149 and 150:
Expected results with competitive e
Page 151 and 152:
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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