AffinityChromatographyPrinciples and Methods18-1022-29Edition AC

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Affinity ChromatographyPrinciples and Methods1

ContentIntroduction . 7Symbols and abbreviations . 8Chapter 1Affinity chromatography in brief . 9BioProcess Media for large-scale production . 12Custom Designed Media and Columns . 12Common terms in affinity chromatography . 13Chapter 2Affinity chromatography in practice . 15Purification steps . 15Media selection . 16Preparation of media and buffers . 16Sample preparation and application . 17Elution . 18Flow rates . 21Analysis of results and further steps . 21Equipment selection . 21Troubleshooting . 22Chapter 3Purification of specific groups of molecules . 25Immunoglobulins . 25IgG, IgG fragments and subclasses . 26HiTrap Protein G HP, Protein G Sepharose 4 Fast Flow, MAbTrap Kit . 28HiTrap Protein A HP, Protein A Sepharose 4 Fast Flow, HiTrap rProtein A FF,rProtein A Sepharose 4 Fast Flow . 33Monoclonal IgM from hybridoma cell culture . 38HiTrap IgM Purification HP . 38Avian IgY from egg yolk . 40HiTrap IgY Purification HP . 40Recombinant fusion proteins . 42GST fusion proteins . 42GST MicroSpin Purification Module, GSTrap FF, Glutathione Sepharose 4 Fast Flow,Glutathione Sepharose 4B . 42Poly (His) fusion proteins . 46His MicroSpin Purification Module, HisTrap Kit, HiTrap Chelating HP,Chelating Sepharose Fast Flow . 46Protein A fusion proteins . 51IgG Sepharose 6 Fast Flow . 51Purification or removal of serine proteases,e.g. thrombin and trypsin, and zymogens . 53HiTrap Benzamidine FF (high sub), Benzamidine Sepharose 4 Fast Flow (high sub) . 532

Serine proteases and zymogens with an affinity for arginine . 57Arginine Sepharose 4B . 57DNA binding proteins . 59HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow . 59Coagulation factors . 64HiTrap Heparin HP, HiPrep 16/10 Heparin FF, Heparin Sepharose 6 Fast Flow . 64Biotin and biotinylated substances . 65HiTrap Streptavidin HP, Streptavidin Sepharose High Performance . 65Purification or removal of fibronectin . 68Gelatin Sepharose 4B . 68Purification or removal of albumin . 69HiTrap Blue HP, Blue Sepharose 6 Fast Flow . 69NAD -dependent dehydrogenases and ATP-dependent kinases . 725' AMP Sepharose 4B, HiTrap Blue HP, Blue Sepharose 6 Fast Flow . 72NADP -dependent dehydrogenases and other enzymes with affinity for NADP . 742'5' ADP Sepharose 4B, Red Sepharose CL-6B . 74Glycoproteins or polysaccharides . 79Con A Sepharose 4B, Lentil Lectin Sepharose 4B, Agarose Wheat Germ Lectin . 79Con A for binding of branched mannoses, carbohydrates with terminal mannoseor glucose (aMan aGlc GlcNAc) . 79Lentil lectin for binding of branched mannoses with fucose linked a(1,6) tothe N-acetyl-glucosamine, (aMan aGlc GlcNAc) N-acetylglucosaminebinding lectins . 82Wheat germ lectin for binding of chitobiose core of N-linked oligosaccharides,[GlcNAc(b1,4GlcNAc)1-2 b GlcNAc] . 83Calmodulin binding proteins: ATPases, adenylate cyclases,protein kinases, phosphodiesterases, neurotransmitters . 85Calmodulin Sepharose 4B . 85Proteins 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) . 87HiTrap Chelating HP, Chelating Sepharose Fast Flow,His MicroSpin Purification Module, HisTrap Kit . 87Thiol-containing substances (purification by covalent chromatography) . 91Activated Thiol Sepharose 4B, Thiopropyl Sepharose 6B . 91Chapter 4Components of an affinity medium . 96The matrix . 96The ligand . 97Spacer arms . 98Ligand coupling . 99Ligand specificity . 993

Chapter 5Designing affinity media using pre-activated matrices . 100Choosing the matrix . 100Choosing the ligand and spacer arm . 100Choosing the coupling method . 100Coupling the ligand . 102Binding capacity, ligand density and coupling efficiency . 103Binding and elution conditions . 104Coupling through the primary amine of a ligand . 105HiTrap NHS-activated HP, NHS-activated Sepharose 4 Fast Flow . 105CNBr-activated Sepharose . 108Immunoaffinity chromatography . 112Coupling small ligands through amino or carboxyl groupsvia a spacer arm . 113EAH Sepharose 4B and ECH Sepharose 4B . 113Coupling through hydroxy, amino or thiol groupsvia a 12-carbon spacer arm . 116Epoxy-activated Sepharose 6B . 116Coupling through a thiol group . 120Thiopropyl Sepharose 6B . 120Coupling other functional groups . 121Chapter 6Affinity chromatography and CIPP . 123Applying CIPP . 124Selection and combination of purification techniques . 124Appendix 1 . 129Sample preparation . 129Sample stability . 129Sample clarification . 130Specific sample preparation steps . 131Resolubilization of protein precipit