为了方便更多的研究人员系统的学习和了解蛋白纯化技术，生物库会在随后的时间内陆续的推出GE公司蛋白纯化相关技术手册免费提供下载，由于中文手册文件较大，如需中文版技术手册请注册本站评论回复告知邮箱，或点击关注本站新浪微博后发私信给生物库告知您的邮箱，生物库会第一时间Email给您！同时也感谢您对本站的支持与关注！

GE技术手册发布计划：

第一期：
Ion Exchange Chromatography & Chromatofocusing Principles and Methods
离子交换色谱及色谱聚焦原理和方法

第二期：
Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods
疏水相互作用和反向层析技术原理和方法

第三期：
Gel Filtration Principles and Methods
凝胶过滤原理和方法

第四期：
Affinity Chromatography Principles and Methods
亲和色谱原理和方法

第五期：
Antibody Purification Handbook
抗体纯化手册

第六期：
Cell Separation Media Methodology and applications
细胞分离介质方法学和应用

第七期：
Expanded bed adsorption Principles and Methods

第八期：
Protein Purification Handbook
蛋白质纯化手册

第九期：
Microcarrier Cell Culture Principles and Methods

第十期：
Recombinant Protein Purification Handbook Principles and Methods
重组蛋白纯化手册原理和方法

第十一期:
Isolation of mononuclear cells Methodology and applications
单核细胞的分离方法与应用

第十二期：
GST Gene Fusion System Handbook

第十三期：
Purifying Challenging Proteins Principles and Methods
富有挑战性的蛋白质纯化原理和方法

第十四期：
2-D Electrophoresis Principles and Methods
2-D双向电泳的原理与方法

今天我们就发布第四期：
Affinity Chromatography Principles and Methods
亲和色谱原理和方法

英文版：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...............................................................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.................................................................................................................................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
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

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中文版：亲和色谱原理和方法

如需中文版技术手册请注册本站评论回复告知邮箱，或点击关注本站新浪微博后发私信给我告知您的邮箱，我会第一时间Email给您！同时也感谢您对本站的支持与关注！