Affinity Chromatography - Department of Molecular and Cellular ...

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Product Quantity Code No. Con A Sepharose 4B 5 ml 17-0440-03 100 ml 17-0440-01 Gelatin Sepharose 4B 25 ml 17-0956-01 Glutathione Sepharose 4 FF 25 ml 17-5132-01 100 ml 17-0532-02 500 ml 17-0532-03 Glutathione Sepharose 4B 10 ml 17-0756-01 Heparin Sepharose 6 FF 50 ml 17-0998-01 250 ml 17-0998-25 IgG Sepharose 6 FF 10 ml 17-0969-01 Lentil Lectin Sepharose 4B 25 ml 17-0444-01 Red Sepharose CL-6B 10 g 17-0528-01 Streptavidin Sepharose HP 5 ml 17-5113-01 Pre-activated media and columns for ligand coupling HiTrap NHS-activated HP 5 x 1 ml 17-0716-01 1 x 5 ml 17-0717-01 NHS-activated Sepharose 4 FF 25 ml 17-0906-01 CNBr-activated Sepharose 4 FF 10 g 17-0981-01 CNBr-activated Sepharose 4B 15 g 17-0430-01 Activated CH Sepharose 4B 15 g 17-0490-01 ECH Sepharose 4B 50 ml 17-0571-01 Epoxy-activated Sepharose 6B 15 g 17-0840-01 EAH Sepharose 4B 50 ml 17-0569-01 Activated Thiol Sepharose 4B 15 g 17-0640-01 Thiopropyl Sepharose 6B 15 g 17-0420-01 Immunoprecipitation Immunoprecipitation Starter Pack 2 x 2 ml 17-6002-35 Protein A Sepharose 4 Fast Flow Protein G Sepharose 4 Fast Flow 156
STREAMLINE, Sepharose, HiTrap, ÄKTA, MAbTrap, HisTrap, GSTrap, BioProcess, PhastSystem, PhastGel, FPLC, MicroSpin, Microplex, Multiphor, HiPrep, Sephadex, BioDirectory, Hybond, ECL, ECL Plus, ExcelGel and Superdex are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries. Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia Corporation BIACORE is a trademark of Biacore AB Multipipette and Eppendorf are trademarks of Eppendorf-Netheler-Hinz GmbH Tween is a trademark of ICI Americas Inc Cibacron is a registered trademark of Ciba-Geigy Corp Procion and Coomassie are trademarks of ICI plc. Triton is a trademark of Union Carbide Chemicals and Plastics Co. Nonidet is a trademark of Shell Co. Ltd. Pefabloc is a trademark of Pentafam AG. All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group that supplies them. A copy of these terms and conditions is available on request. © Amersham Pharmacia Biotech AB 2001 – All rights reserved. Amersham Pharmacia Biotech AB Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Pharmacia Biotech UK Limited Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Amersham Pharmacia Biotech Inc 800 Centennial Avenue, PO Box 1327, Piscataway, NJ 08855, USA Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9, D-79111 Freiburg, Germany Amersham Pharmacia Biotech KK, Sanken Bldg. 3-25-1, Hyakunincho Shinjuku-ku, Tokyo 169-0073, Japan
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 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
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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
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Sample: Column: Binding buffer: Elu
Page 65 and 66:
Media characteristics Ligand densit
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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
Page 75 and 76:
Purification options Binding capaci
Page 77 and 78:
Sepharose NH (CH 2) n NH N N O N N
Page 79 and 80:
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
Page 89 and 90:
Proteins and peptides with exposed
Page 91 and 92:
Several methods can be used to dete
Page 93 and 94:
Media characteristics Composition M
Page 95 and 96:
Performing a separation Binding buf
Page 97 and 98:
Do not store the suspension for lon
Page 99 and 100:
Sepharose has been modified and dev
Page 101 and 102:
Ligand coupling Several methods are
Page 103 and 104:
Couple a ligand through the least c
Page 105 and 106:
Table 9 summarizes recommended liga
Page 107 and 108: Coupling through the primary amine
Page 109 and 110: Ligand and column preparation 1. Di
Page 111 and 112: Options Product Spacer Coupling con
Page 113 and 114: 100 80 Coupled substance, % 60 40 2
Page 115 and 116: Coupling small ligands through amin
Page 117 and 118: Ligand coupling 1. Add the ligand s
Page 119 and 120: Alternative coupling solutions: Dis
Page 121 and 122: 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
Page 133 and 134: Remove salts from proteins with mol
Page 135 and 136: 1. Filter (0.45 µm) or centrifuge
Page 137 and 138: For laboratory scale operations, Ta
Page 139 and 140: Removal of lipoproteins Lipoprotein
Page 141 and 142: Appendix 2 Selection of purificatio
Page 143 and 144: 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: Ordering information Product Quanti
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