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Additional reading and reference material Code No. Purification Antibody Purification Handbook 18-1037-46 Protein Purification Handbook 18-1132-29 Recombinant Protein Handbook: Protein Amplification and Simple Purification 18-1142-75 Gel Filtration Handbook: Principles and Methods 18-1022-18 Ion Exchange Chromatography Handbook: Principles and Methods 18-1114-21 Hydrophobic Interaction Chromatography Handbook: Principles and Methods 18-1020-90 Reversed Phase Chromatography Handbook: Principles and Methods 18-1112-93 Expanded Bed Adsorption Handbook: Principles and Methods 18-1124-26 Protein and Peptide Purification Technique Selection 18-1128-63 Fast Desalting and Buffer Exchange of Proteins and Peptides 18-1128-62 Gel Filtration Columns and Media Selection Guide 18-1124-19 Ion Exchange Columns and Media Selection Guide 18-1127-31 HIC Columns and Media Product Profile 18-1100-98 Affinity Columns and Media Product Profile 18-1121-86 Convenient Protein Purification, HiTrap Column Guide 18-1128-81 ÄKTAdesign Brochure 18-1129-05 Column Packing Video (PAL) 17-0893-01 Column Packing Video (NTSC) 17-0894-01 Analysis Gel Media Guide (electrophoresis) 18-1129-79 2D Electrophoresis Handbook 80-6429-60 Protein Electrophoresis Technical Manual 80-6013-88 ECL Western and ECL Plus Western Blotting Application Note 18-1139-13 Many of these items can be downloaded from www.apbiotech.com 154
Ordering information Product Quantity Code No. Prepacked columns HiTrap rProtein A FF 2 x 1 ml 17-5079-02 5 x 1 ml 17-5079-01 1 x 5 ml 17-5080-01 HiTrap Protein A HP 2 x 1 ml 17-0402-03 5 x 1 ml 17-0402-01 1 x 5 ml 17-0403-01 HiTrap Protein G HP 2 x 1 ml 17-0404-03 5 x 1 ml 17-0404-01 1 x 5 ml 17-0405-01 HiTrap Blue HP 5 x 1 ml 17-0412-01 1 x 5 ml 17-0413-01 HiTrap Heparin HP 5 x 1 ml 17-0406-01 1 x 5 ml 17-0407-01 HiTrap NHS-activated HP 5 x 1 ml 17-0716-01 1 x 5 ml 17-0717-01 HiTrap Chelating HP 5 x 1 ml 17-0408-01 1 x 5 ml 17-0409-01 HiTrap Streptavidin HP 5 x 1 ml 17-5112-01 HiTrap IgM Purification HP 5 x 1 ml 17-5110-01 HiTrap IgY Purification HP 1 x 5 ml 17-5111-01 GSTrap FF 2 x 1 ml 17-5130-02 5 x 1 ml 17-5130-01 1 x 5 ml 17-5131-01 HiTrap Benzamidine FF (high sub) 2 x 1 ml 17-5143-02 5 x 1 ml 17-5143-01 1 x 5 ml 17-5144-01 HiPrep 16/10 Heparin FF 1 x 20 ml 17-5189-01 Kits MAbTrap Kit HiTrap Protein G HP (1 x 1 ml), 17-1128-01 accessories, pre-made buffers for 10 purifications HisTrap Kit 3 x 1 ml HiTrap Chelating HP 17-1880-01 columns, pre-made buffers and accessories for uo to 12 purifications Media Protein A Sepharose CL-4B 1.5 g 17-0780-01 25 ml 17-0963-03 Protein A Sepharose 4 FF 5 ml 17-0974-01 25 ml 17-0974-04 rProtein A Sepharose FF 5 ml 17-1279-01 25 ml 17-1279-02 Protein G Sepharose 4 FF 5 ml 17-0618-01 25 ml 17-0618-02 2´,5´ ADP Sepharose 4B 5 g 17-0700-01 5´ AMP Sepharose 4B 5 g 17-0620-01 Agarose Wheat Germ Lectin 5 ml 27-3608-02 Arginine Sepharose 4B 25 ml 17-0524-01 Benzamidine Sepharose 4 FF (high sub) 25 ml 17-5123-01 Blue Sepharose 6 FF 50 ml 17-0948-01 Calmodulin Sepharose 4B 10 ml 17-0529-01 Chelating Sepharose FF 50 ml 17-0575-01 155
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
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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
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Table 2. Relative binding strengths
Page 31 and 32:
Purification examples Figure 11 sho
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MAbTrap Kit Fig. 13. MAbTrap Kit, r
Page 35 and 36:
Media characteristics Ligand Compos
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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
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in The Recombinant Protein Handbook
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Cleaning These procedures are appli
Page 49 and 50:
Purification examples Figures 24 an
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Nickel solution: 0.1 M NiSO 4 Bindi
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Protein A fusion proteins IgG Sepha
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Purification or removal of serine p
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Sample: 2 ml clarified E. coli homo
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Serine proteases and zymogens with
Page 61 and 62:
DNA binding proteins HiTrap Heparin
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Sample: Column: Binding buffer: Elu
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Media characteristics Ligand densit
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Sample: Pooled and frozen human pla
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The harsh conditions required to br
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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
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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
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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
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: Appendix 9 Storage of biological sa
Page 159 and 160: STREAMLINE, Sepharose, HiTrap, ÄKT
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