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Affinity Chromatography Principles and Methods GE Healthcare GE GE Healthcare Affinity Chromatograpy Handbook – Principles and Methods GE, imagination at work and GE Monogram are trademarks of General Electric Company. ÄKTA, ÄKTAexplorer, ÄKTAFLPC, ÄKTAprime, ÄKTApurifier, Biacore, BioDirectory, BioProcess, ECL, ECL Plus, ExcelGel, FPLC, GSTPrep, GSTrap, HisTrap, HiPrep, HiTrap, Hybond, MAbTrap, MabSelect, MicroSpin, Microplex, Multiphor, STREAMLINE, Sepharose, Percoll, PhastSystem, PhastGel, Sephadex, Superdex, and Tricorn are trademarks of GE Healthcare companies. Purification and preparation of fusion proteins and affinity peptides comprising at least two adjacent histidine residues may require a license under US pat 5,284,933 and US pat 5,310,663, including corresponding foreign patents (assigne: Hoffman La Roche, Inc). A license for commercial use of GST gene fusion vectors must be obtained from Chemicon International, Incorprated, 28820 Singel Oak Drive, Temecula, California 92590 USA. The Tricorn column and components are protected by US design patents USD500856, USD506261, USD500555, USD495060 and their equivalents in other countries. All third party trademarks are the property of their respective owners. © 1988–2007 General Electric Company – All rights reserved. Affinity Chromatography First published 1988. All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare that supplies them. A copy of these terms and Principles and Methods For local office contact information, conditions is available on request. Contact your local GE Healthcare representative please visit www.gelifesciences.com/contact for the most current information. GE Healthcare Europe GmbH Munzinger Strasse 5 GE Healthcare Bio-Sciences AB D-79111 Freiburg, Germany GE Healthcare UK Limited Björkgatan 30 Amersham Place Little Chalfont 751 84 Uppsala Buckinghamshire, HP7 9NA, UK Sweden GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue P.O. Box 1327 www.gelifesciences.com/protein-purification Piscataway, NJ 08855-1327, USA GE Healthcare Bio-Sciences KK Sanken Bldg.3-25-1 Hyakunincho Shinjuku-ku Tokyo 169-0073, Japan 18-1022-29 AE 10/2007 18-1022-29 AE Cover GE.indd 2 10/15/2007 16:52:27 Handbooks from GE Healthcare Protein Purification GST Gene Fusion System Handbook Handbook 18-1132-29 18-1157-58 Gel Filtration Hydrophobic Interaction and Principles and Methods Reversed Phase Chromatography 18-1022-18 Principles and Methods 11-0012-69 Affinity Chromatography Principles and Methods 2-D Electrophoresis 18-1022-29 using immobilized pH gradients Principles and Methods Antibody Purification 80-6429-60 Handbook 18-1037-46 Microcarrier Cell Culture Principles and Methods Percoll 18-1140-62 Methodology and Applications 18-1115-69 Challenging Protein Purification Handbook Ion Exchange Chromatography 28-9095-31 & Chromatofocusing Principles and Methods Recombinant Protein 11-0004-21 Purification Handbook Principles and Methods Purifying Challenging Proteins 18-1142-75 Principles and Methods 28-9095-31 18-1022-29 AE Cover GE.indd 3 10/15/2007 16:52:28 Affinity Chromatography Principles and Methods Contents Introduction ............................................................................................................. 7 Symbols and abbreviations ............................................................................................................................8 Chapter 1 Affinity 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 Affinity 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
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