Affinity Chromatography – Affinity Chromatography Principles and Methods Production: RAK Design AB Principles and Methods www.chromatography.amershambiosciences.com 18-1022-29 Edition AD Handbooks from Amersham Biosciences STREAMLINE, Sepharose, HiTrap, ÄKTA, MAbTrap, HisTrap, GSTrap, BioProcess, PhastSystem, PhastGel, FPLC, MicroSpin, Microplex, Multiphor, HiPrep, Sephadex, BioDirectory, Hybond, ECL, ECL Plus, ExcelGel, Antibody Purification Superdex, GSTPrep, MabSelect, Tricorn and Drop Design are trademarks of Amersham Biosciences Limited. Handbook 18-1037-46 Amersham and Amersham Biosciences are trademarks of Amersham plc. BIACORE is a trademark of Biacore AB. The Recombinant Protein Handbook Gel Filtration Multipipette and Eppendorf are trademarks of Eppendorf-Netheler-Hinz GmbH. Tween is a trademark of ICI Americas Inc. 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Principles and Methods with Polybuffer and PBE 18-1157-58 18-1022-29 18-1009-07 Amersham Biosciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden 2-D Electrophoresis Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Hydrophobic Interaction Chromatography Microcarrier cell culture using immobilized pH gradients Amersham Biosciences Corp., 800 Centennial Avenue, PO Box 1327, Piscataway NJ 08855, USA Principles and Methods Principles and Methods Principles and Methods Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111 Freiburg, Germany 18-1020-90 18-1140-62 80-6429-60 Amersham Biosciences K.K., Sanken Bldg. 3-25-1, Hyakunincho Amersham Shinjuku-ku, Tokyo 169-0073, Japan Affinity Chromatography Principles and Methods 1 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 2 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 ............................................................. 73 5' AMP Sepharose 4B .............................................................................................................................. 74 HiTrap Blue HP, Blue Sepharose 6 Fast Flow