《食品分析》课程实验室操作参考手册（Amersham Biosciences）05 亲和层析手册 Affinity Chromatography Principles and Methods

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

Chapter 5 Designing affinity media using pre-activated matrices. .101 Choosing the matrix. .10 ng m 103 Binding capacity,ligand density and coupling 104 Binding and elution conditions. 105 Coupling through the primary amine of a ligand. 106 HiTrap NHS-activated HP.NHS-activated Sepharose 4 Fast Flow CNBr-actrvated Sepharose . d ECH Sep 17 coupingthoughathiolgroup 1 Couplingother Chapter 6 Affinity chromatography and C 123 Applying CF 124 on and combination of purification techniques 124 128 pe preparation 128 29 -12 Specific sample r preparation steps 30 133 Buffer exchange and desalting 133 Removal of lin 136 Removal ofp phenolred 136 Removal of low molecular weight contaminants 136 137 Appendix3 Column packing and preparation 138

4 Chapter 5 Designing affinity media using pre-activated matrices . 101 Choosing the matrix .101 Choosing the ligand and spacer arm . 101 Choosing the coupling method .101 Coupling the ligand . 103 Binding capacity, ligand density and coupling efficiency .104 Binding and elution conditions .105 Coupling through the primary amine of a ligand . 106 HiTrap NHS-activated HP, NHS-activated Sepharose 4 Fast Flow .106 CNBr-activated Sepharose .109 Immunoaffinity chromatography . 113 Coupling small ligands through amino or carboxyl groups via a spacer arm . 114 EAH Sepharose 4B and ECH Sepharose 4B .114 Coupling through hydroxy, amino or thiol groups via a 12-carbon spacer arm . 117 Epoxy-activated Sepharose 6B .117 Coupling through a thiol group . 121 Thiopropyl Sepharose 6B . 121 Coupling other functional groups . 122 Chapter 6 Affinity chromatography and CIPP . 123 Applying CIPP . 124 Selection and combination of purification techniques . 124 Appendix 1 . 128 Sample preparation . 128 Sample stability .128 Sample clarification .129 Specific sample preparation steps . 130 Resolubilization of protein precipitates .133 Buffer exchange and desalting . 133 Removal of lipoproteins. 136 Removal of phenol red . 136 Removal of low molecular weight contaminants . 136 Appendix 2 . 137 Selection of purification equipment . 137 Appendix 3 . 138 Column packing and preparation . 138

Appendix 4 140 om)to 140 Appendix 5. 141 co data:proteins.column pressures 1A1 .141 Appendix 6. .142 Table of amino acids. 142 Appendix 7. .144 Kinetics in affinity chromatography .144 Appendix 8. .149 Analytical assays during purification 149 Appendix 9. .151 Storage of biological samples 151 Product index. 152 Additional reading 153 References. 153 Ordering infomation 154

5 Appendix 4 . 140 Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa . 140 Appendix 5 . 141 Conversion data: proteins, column pressures . 141 Column pressures . 141 Appendix 6 . 142 Table of amino acids . 142 Appendix 7 . 144 Kinetics in affinity chromatography . 144 Appendix 8 . 149 Analytical assays during purification . 149 Appendix 9 . 151 Storage of biological samples . 151 Product index . 152 Additional reading . 153 References . 153 Ordering information . 154

Introduction Biomolecules are purified using purification techniques that separate according todifferences in specific properties,as shown in Figure 1. Property Technique Biorecoition (ligand specificity) Affinity chromatography Charge on exchange chromatography Hydrophobicity Heeeaoncwoagrapw 从> Gel filtration Hydrophobic interaction Ion exchange Reversed phas Fig.1.Separation principles in chromatographic purification. Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins)and a specific ligand coupled to a chromatography matrix. The technique offers high selectivity,hence high resolution,and usually high ca pacity for the protein(s)of interest.Purification can be in the order of several thousand-fold and recoveries of active material are generally very high. that the pu cation ation chromatography.The technique can be used to separate active biomolecules from denatured or functionally different forms,to isolate pure substances present at low concentration in large volumes of crude sample and also to remove specific contaminants. Amersham Biosciences offers a wide variety of prepacked columns,ready to use media,and pre-activated media for ligand coupling

7 Introduction Biomolecules are purified using purification techniques that separate according to differences in specific properties, as shown in Figure 1. Property Technique Biorecognition (ligand specificity) Affinity chromatography Charge Ion exchange chromatography Size Gel filtration (sometimes called size exclusion) Hydrophobicity Hydrophobic interaction chromatography Reversed phase chromatography Fig. 1. Separation principles in chromatographic purification. Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatography matrix. The technique offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest. Purification can be in the order of several thousand-fold and recoveries of active material are generally very high. Affinity chromatography is unique in purification technology since it is the only technique that enables the purification of a biomolecule on the basis of its biological function or individual chemical structure. Purification that would otherwise be time-consuming, difficult or even impossible using other techniques can often be easily achieved with affinity chromatography. The technique can be used to separate active biomolecules from denatured or functionally different forms, to isolate pure substances present at low concentration in large volumes of crude sample and also to remove specific contaminants. Amersham Biosciences offers a wide variety of prepacked columns, ready to use media, and pre-activated media for ligand coupling. Gel filtration Hydrophobic interaction Ion exchange Affinity Reversed phase

This handbook describes the role of affinity chromatography in the purification of biomolecules,the principle of the technique,the media available and how to select them, 26plesandkuikdnsnmtisortcmo以commoctomedpoot ation is given as a guide towards obtaining the best results. The lstration on the inside cove hows the range of handbooks that have been produced ersham e that ith any chrom mleand dureycale ndaai igu Symbols and abbreviations this symbol indicates general advice which can improve procedures or provide recommendations for action under specific situations. this symbol denotes advice which should be regarded as mandatory and gives a warning when special care should be taken. this symbol highlights troubleshooting advice to help analyze and resolve difficulties that may occur. chemicals,buffers and equipment. experimental protocol. PBS phosphate buffered saline (140 mM NaCl,2.7 mM KCl,10 mM Naz HPO 1.8 mM KH2PO4,pH 7.4)

8 This handbook describes the role of affinity chromatography in the purification of biomolecules, the principle of the technique, the media available and how to select them, application examples and detailed instructions for the most commonly performed procedures. Practical information is given as a guide towards obtaining the best results. The illustration on the inside cover shows the range of handbooks that have been produced by Amersham Biosciences to ensure that purification with any chromatographic technique becomes a simple and efficient procedure at any scale and in any laboratory. Symbols and abbreviations this symbol indicates general advice which can improve procedures or provide recommendations for action under specific situations. this symbol denotes advice which should be regarded as mandatory and gives a warning when special care should be taken. this symbol highlights troubleshooting advice to help analyze and resolve difficulties that may occur. chemicals, buffers and equipment. experimental protocol. PBS phosphate buffered saline (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4)

Chapter 1 Affinity chromatography in brief Affinity chromatography separaes n the basis of reversibleinteraction between a protein(or group of proteins)and a specific ligand coupled to a chromatography matrix The technique is ideal for a capture or intermediate step in a purification protocol and can be used whenever a suitable ligand is available for the protein(s)of interest.With high selectivity, hence high resolution,and high capacity for the protein(s)of interest,purification levels in the order of several thousand-fold with high recovery of active material are achievable. Target protein(s)is collected in a purified,concentrated form. Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic interactions.van der waals'forces and/or hydrogen bonding.To elute the target molecule from the affinity medium the interaction can be reversed,either specifically using a competitive ligand,or non-specifically,by changing the pH,ionic strength or polarity. In a single step,affinity purification can offer immense time-saving over less selective multi- nables plex biolog processed.Targe rom com ame substance ar For an even higher degree of purity,or when there is no suitable ligand for affinity purification, an efficient multi-step process must be developed using the purification strategy of Capture, Intermediate Purification and Polishing(Cp).When applying this strategy affinity chroma- tography offers an ideal capture or intermediate step in any purification protocol and can be used whenever a suitable ligand is available for the protein of interest. Successful affinity purification requires a biospecific ligand that can be covalently attached to a chromatography matrix.The coupled ligand must retain its specific binding affinity for the target molecules and,after washing away unbound material,the binding between the ligand and target molecule must be reversible to allow the target molecules to be removed in an active form.Any comp onent can be used as a ligand to ourify its respective binding .Enzymesubstrate analogue,inhibitor,cofactor. ·Antibody÷antigen,virus,cel. Lectin polysaccharide,glycoprotein,cell surface receptor,cell .Nucleic acidcomplementary base sequence,histones,nucleic acid polymerase, nucleic acid binding protein. Hormone,vitamin receptor,carrier protein. .Glutathioneglutathione-S-transferase or GST fusion proteins. .Metal ions Poly(His)fusion proteins,native proteins with histidine,cysteine and/or tryptophan residues on their surfaces

9 Chapter 1 Affinity chromatography in brief Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatography matrix. The technique is ideal for a capture or intermediate step in a purification protocol and can be used whenever a suitable ligand is available for the protein(s) of interest. With high selectivity, hence high resolution, and high capacity for the protein(s) of interest, purification levels in the order of several thousand-fold with high recovery of active material are achievable. Target protein(s) is collected in a purified, concentrated form. Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. To elute the target molecule from the affinity medium the interaction can be reversed, either specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. In a single step, affinity purification can offer immense time-saving over less selective multi￾step procedures. The concentrating effect enables large volumes to be processed. Target molecules can be purified from complex biological mixtures, native forms can be separated from denatured forms of the same substance and small amounts of biological material can be purified from high levels of contaminating substances. For an even higher degree of purity, or when there is no suitable ligand for affinity purification, an efficient multi-step process must be developed using the purification strategy of Capture, Intermediate Purification and Polishing (CIPP). When applying this strategy affinity chroma￾tography offers an ideal capture or intermediate step in any purification protocol and can be used whenever a suitable ligand is available for the protein of interest. Successful affinity purification requires a biospecific ligand that can be covalently attached to a chromatography matrix. The coupled ligand must retain its specific binding affinity for the target molecules and, after washing away unbound material, the binding between the ligand and target molecule must be reversible to allow the target molecules to be removed in an active form. Any component can be used as a ligand to purify its respective binding partner. Some typical biological interactions, frequently used in affinity chromatography, are listed below: • Enzyme
substrate analogue, inhibitor, cofactor. • Antibody
antigen, virus, cell. • Lectin
polysaccharide, glycoprotein, cell surface receptor, cell. • Nucleic acid
complementary base sequence, histones, nucleic acid polymerase, nucleic acid binding protein. • Hormone, vitamin
receptor, carrier protein. • Glutathione
glutathione-S-transferase or GST fusion proteins. • Metal ions
Poly (His) fusion proteins, native proteins with histidine, cysteine and/or tryptophan residues on their surfaces