Affinity Chromatography Principles and Methods Amersham 18-1022-29 Biosciences Edition AD
18-1022-29 Edition AD Affinity Chromatography – Principles and Methods Production: RAK Design AB Principles and Methods Affinity Chromatography www.chromatography.amershambiosciences.com
Handbooks from Amersham Biosciences Antibody Purification 18-103746 The Recombinant Protein Handbook nd Simple Purification 18-102 Methods Percoll Protein Purification Reversed Phase Chr atography 1819229 d Methods nlicin Ficoll-Paque Plus 1g-1h52golatonoftmphocgtes GST Gene Fusion System chromatofocusing 1g5758 Hydrophobic Interaction Chromatography Microcarrier cell culture
Percoll Methodology and Applications 18-1115-69 Ficoll-Paque Plus For in vitro isolation of lymphocytes 18-1152-69 GST Gene Fusion System Handbook 18-1157-58 2-D Electrophoresis using immobilized pH gradients Principles and Methods 80-6429-60 Antibody Purification Handbook 18-1037-46 The Recombinant Protein Handbook Protein Amplification and Simple Purification 18-1142-75 Protein Purification Handbook 18-1132-29 Ion Exchange Chromatography Principles and Methods 18-1114-21 Affinity Chromatography Principles and Methods 18-1022-29 Hydrophobic Interaction Chromatography Principles and Methods 18-1020-90 Gel Filtration Principles and Methods 18-1022-18 Reversed Phase Chromatography Principles and Methods 18-1134-16 Expanded Bed Adsorption Principles and Methods 18-1124-26 Chromatofocusing with Polybuffer and PBE 18-1009-07 Microcarrier cell culture Principles and Methods 18-1140-62 STREAMLINE, Sepharose, HiTrap, ÄKTA, MAbTrap, HisTrap, GSTrap, BioProcess, PhastSystem, PhastGel, FPLC, MicroSpin, Microplex, Multiphor, HiPrep, Sephadex, BioDirectory, Hybond, ECL, ECL Plus, ExcelGel, Superdex, GSTPrep, MabSelect, Tricorn and Drop Design are trademarks of Amersham Biosciences Limited. Amersham and Amersham Biosciences are trademarks of Amersham plc. 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. 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). All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them. A copy of these terms and conditions is available on request. © Amersham Biosciences AB 2002 – All rights reserved. Amersham Biosciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden Amersham Biosciences UK Limited, Amersham Place, Little Chalfont, Buckinghamshire HP7 9NA, England Amersham Biosciences Corp., 800 Centennial Avenue, PO Box 1327, Piscataway NJ 08855, USA Amersham Biosciences Europe GmbH, Munzinger Strasse 9, D-79111 Freiburg, Germany Amersham Biosciences K.K., Sanken Bldg. 3-25-1, Hyakunincho Amersham Shinjuku-ku, Tokyo 169-0073, Japan Handbooks from Amersham Biosciences
Affinity Chromatography Principles and Methods
1 Affinity Chromatography Principles and Methods
Contents Introduction. 7 Symbols and abbreviations matography in brief BioProcess Media for large-scale production Custom Designed Media and Columns . 12 Common terms in affinity chromatography . .13 Chapter 2 Affinity chromatography in practice. steps 5 1 Prenaration of media and hutfers 1 Sample preparation and applicatior 17 lution and further steps 2 .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 sForap ProtinAFE. 33 Monoclonal IgM from hybridoma cell culture. 38 HiTran leM Putification HP 38 Avian IgY from egg yolk. 40 HiTtan leY Purification HP AO Recombinant fusion proteins. 42 GST fusion proteins .42 Poly(His)fusion proteins Protein A fusion proteins. 5 gGSepharose6 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 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 Bonandb rep 16/10 Heparin FF.Heparin Sepharose 6 Fast Flow otinylated substances. n Sepharose High Performance n o 69 Purification or oval of albumin 70 nd ATP-dependent kinase 73 lue HP.Blue Sep 6 Fast Flow 74 HiTrap Blue HP Blue Sepharose6 Fast Flow. 75 NADP-dependent dehydrogenases and other enzymes with affinity for NADP. 75 2'5'ADP Sepharose 4B.Red Sepharose CL-6B. 25'ADP Sepharose 4B Red Seph se CL-68 Glycoproteins or polysa charides Lentil Lectin Sepharos 0 83 Ge是BTegnAbindgCciobosecoreotliakedoleosachaides, 84 Calmodulin binding proteins:ATPases.adenvlate cyclases.protein kinases. phosphodiesterases,neurotransmitters. .86 Calmodulin Sepharose 4B . -86 Proteins and peptides wit immobilized metal chelate affinity chromatographv). .88 HiTrap Chelating HP.Chelating Sepharose Fast Flow,His MicroSpin Purification Module.HisTrap Kit8 Thiol-containing substances (purification by covalent chromatography). 92 Activated Thiol Sepharose 4B.Thiopropyl Sepharose 6B . .92 Chapter 4 Components of an affinity medium 9 The matro 90 100 Ligand specificity 100
3 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 .75 NADP+-dependent dehydrogenases and other enzymes with affinity for NADP+ . 75 2'5' ADP Sepharose 4B, Red Sepharose CL-6B .75 2'5' ADP Sepharose 4B .77 Red Sepharose CL-6B . 78 Glycoproteins or polysaccharides . 80 Con A Sepharose 4B, Lentil Lectin Sepharose 4B, Agarose Wheat Germ Lectin .80 Con A for binding of branched mannoses, carbohydrates with terminal mannose or glucose (aMan > aGlc > GlcNAc) .80 Lentil lectin for binding of branched mannoses with fucose linked a(1,6) to the N-acetyl-glucosamine, (aMan > aGlc > GlcNAc) N-acetylglucosamine binding lectins . 83 Wheat germ lectin for binding of chitobiose core of N-linked oligosaccharides, [GlcNAc(b1,4GlcNAc)1-2 > b GlcNAc] . 84 Calmodulin binding proteins: ATPases, adenylate cyclases, protein kinases, . phosphodiesterases, neurotransmitters . 86 Calmodulin Sepharose 4B . 86 Proteins and peptides with exposed amino acids: His, Cys, Trp, and/or with affinity for metal ions (also known as IMAC, immobilized metal chelate affinity chromatography) . 88 HiTrap Chelating HP, Chelating Sepharose Fast Flow, His MicroSpin Purification Module, HisTrap Kit . 88 Thiol-containing substances (purification by covalent chromatography) . 92 Activated Thiol Sepharose 4B, Thiopropyl Sepharose 6B . 92 Chapter 4 Components of an affinity medium . 97 The matrix .97 The ligand. 98 Spacer arms . 99 Ligand coupling . 100 Ligand specificity . 100