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

英文版：Ion Exchange Chromatography & Chromatofocusing Principles and Methods

Contents
Introduction ............................................................................................................. 7
Symbols .................................................................................................................................................. 8
Common abbreviations .............................................................................................................................. 9
Chapter 1
Principles of ion exchange ..................................................................................... 11
Net surface charge and pH ......................................................................................... 11
Steps in an IEX separation ......................................................................................... 12
Resolution ................................................................................................................ 14
Efficiency ..............................................................................................................................................15
Selectivity ..............................................................................................................................................17
Components of ion exchange media ............................................................................ 21
Matrix ....................................................................................................................................................21
Functional groups ...................................................................................................................................23
Binding capacity and recovery .................................................................................... 25
Chapter 2
Ion exchange in practice ........................................................................................ 26
Introduction .............................................................................................................. 26
Media selection ......................................................................................................... 26
Capture .................................................................................................................................................26
Intermediate purification ..........................................................................................................................27
Polishing ...............................................................................................................................................27
Fast media selection and method development ............................................................. 30
Automated media selection, method development and optimization ..............................................................30
Manual media selection, method development and optimization ...................................................................31
Practical considerations for IEX separation ................................................................... 34
pH and ionic strength ...............................................................................................................................34
Anion or cation exchanger ........................................................................................................................35
Strong or weak ion exchangers ..................................................................................................................36
Buffer selection and preparation ...............................................................................................................37
Column and media preparation .................................................................................................................39
Sample preparation .................................................................................................................................39
Sample application .................................................................................................................................40
Sample load ...........................................................................................................................................40
Elution ..................................................................................................................................................42
Linear gradient elution .............................................................................................................................42
Step elution ...........................................................................................................................................44
pH elution ..............................................................................................................................................46
Flow rates ..............................................................................................................................................46
Flow control ...........................................................................................................................................47
Wash and re-equilibration .........................................................................................................................48
Detergents, denaturing agents and other additives ......................................................................................48
Analysis of results and further steps ............................................................................ 50
Scaling-up ................................................................................................................ 51
Equipment selection .................................................................................................. 52
Care of ion exchange media ........................................................................................ 52
Troubleshooting ......................................................................................................... 53
BioProcess Media – made for bioprocessing ................................................................. 58
Custom Designed Media ............................................................................................. 58

Chapter 3
Ion exchange media ............................................................................................... 59
Introduction .............................................................................................................. 59
MiniBeads: purification or analysis of microgram-milligram quantities
with highest resolution ............................................................................................... 60
Purification options .................................................................................................................................61
Purification examples ...............................................................................................................................62
Performing a separation ............................................................................................................................63
Cleaning ................................................................................................................................................65
Media characteristics ...............................................................................................................................65
Chemical stability ................................................................................................................................... 66
Storage .................................................................................................................................................. 66
MonoBeads: purification of milligram quantities with highest resolution .......................... 67
Purification options .................................................................................................................................68
Purification examples ...............................................................................................................................69
Performing a separation ............................................................................................................................71
Cleaning ................................................................................................................................................72
Media characteristics ...............................................................................................................................73
Chemical stability ................................................................................................................................... 73
Storage .................................................................................................................................................. 73
SOURCE: purification at high throughput with high resolution and easy scale-up ............. 74
Purification options .................................................................................................................................75
Purification examples ...............................................................................................................................76
Performing a separation ............................................................................................................................80
Cleaning ................................................................................................................................................82
Media characteristics ...............................................................................................................................82
Chemical stability ................................................................................................................................... 83
Storage .................................................................................................................................................. 83
Sepharose High Performance: purification with high resolution ...................................... 84
Purification options .................................................................................................................................84
Purification examples ...............................................................................................................................86
Scaling-up .............................................................................................................................................86
Performing a separation ............................................................................................................................89
Cleaning ................................................................................................................................................91
Media characteristics ...............................................................................................................................92
Chemical stability ................................................................................................................................... 92
Storage .................................................................................................................................................. 92
Sepharose Fast Flow: purification with good resolution and easy scale-up ........................ 93
Purification options .................................................................................................................................94
Purification examples ...............................................................................................................................97
Performing a separation .......................................................................................................................... 100
Cleaning .............................................................................................................................................. 102
Media characteristics ............................................................................................................................. 103
Chemical stability ................................................................................................................................. 104
Storage ................................................................................................................................................ 104
Sepharose XL – for selected proteins that require very high binding capacity
to increase productivity, easy scale-up ....................................................................... 105
Purification options ............................................................................................................................... 105
Purification examples ............................................................................................................................. 107
Performing a separation .......................................................................................................................... 109
Cleaning .............................................................................................................................................. 111
Media characteristics ............................................................................................................................. 111
Chemical stability .................................................................................................................................112
Storage ................................................................................................................................................112
Sepharose Big Beads: purification from crude, viscous samples at large scale ............... 113
Purification options ............................................................................................................................... 114
Performing a separation .......................................................................................................................... 115
Cleaning .............................................................................................................................................. 116
Media characteristics ............................................................................................................................. 116
Chemical stability .................................................................................................................................117
Storage ................................................................................................................................................117
Chapter 4
Ion exchange in a Purification Strategy (CIPP) ........................................................ 119
Applying CIPP .......................................................................................................... 120
Selection and combination of purification techniques ................................................. 120
Ion exchange as a capture step ................................................................................. 123
Ion exchange for intermediate purification ................................................................. 125
Ion exchange as a polishing step ............................................................................... 127
Alternative techniques for polishing steps .................................................................. 128
Chapter 5
Chromatofocusing: principles and methods ........................................................... 129
Introduction ............................................................................................................ 129
Media selection ....................................................................................................................................132
Buffer selection ....................................................................................................................................133
Purification options ............................................................................................................................... 134
Buffer options ...................................................................................................................................... 134
Purification examples ............................................................................................................................. 134
Packing a column .................................................................................................................................136
Selection of pH gradient and buffers .......................................................................................................137
Buffer preparation ................................................................................................................................ 140
Sample preparation ............................................................................................................................... 141
Performing a separation .......................................................................................................................... 141
Optimization ........................................................................................................................................ 143
Troubleshooting ....................................................................................................................................144
Removing Polybuffer .............................................................................................................................. 148
Cleaning .............................................................................................................................................. 148
Media characteristics ............................................................................................................................. 149
Chemical stability .................................................................................................................................150
Storage ................................................................................................................................................150
Chromatofocusing and CIPP ....................................................................................... 150
Appendix 1 .......................................................................................................... 151
Sample preparation ................................................................................................. 151
Sample stability ....................................................................................................................................151
Sample clarification ...............................................................................................................................152
Specific sample preparation steps ............................................................................. 153
Resolubilization of protein precipitates ....................................................................................................156
Buffer exchange and desalting .................................................................................. 156
Removal of lipoproteins ............................................................................................ 159
Removal of phenol red ............................................................................................. 159
Removal of low molecular weight contaminants .......................................................... 159
Appendix 2 .......................................................................................................... 160
Non-volatile and volatile buffer systems ..................................................................... 160
Non-volatile buffers for anion exchange chromatography ............................................................................ 160
Non-volatile buffers for cation exchange chromatography ........................................................................... 161
Volatile buffer systems ........................................................................................................................... 161
Appendix 3 .......................................................................................................... 162
Column packing and preparation ............................................................................... 162
Column selection .................................................................................................................................. 164
Column packing and efficiency ............................................................................................................... 164
Appendix 4 .......................................................................................................... 167
Selection of purification equipment ........................................................................... 167
Appendix 5 .......................................................................................................... 168
Converting from linear flow (cm/hour) to volumetric flow rates (ml/min)
and vice versa ......................................................................................................... 168
Appendix 6 .......................................................................................................... 169
Conversion data: proteins, column pressures .............................................................. 169
Column pressures ................................................................................................................................. 169
Appendix 7 .......................................................................................................... 170
Table of amino acids ................................................................................................ 170
Appendix 8 .......................................................................................................... 172
Analytical assays during purification .......................................................................... 172
Appendix 9 .......................................................................................................... 174
Storage of biological samples ................................................................................... 174
Appendix 10 ........................................................................................................ 175
Column cleaning ..................................................................................................... 175
Removal of common contaminants .......................................................................................................... 175
Removal of precipitated proteins, lipids, hydrophobically bound proteins or lipoproteins ............................... 175
Additional reading ............................................................................................... 178
References .......................................................................................................... 179
Ordering information ............................................................................................ 180
Ion exchange .......................................................................................................... 180
Chromatofocusing .................................................................................................... 183
Product index ...................................................................................................... 184

更多内容请点击下载：Ion Exchange Chromatography & Chromatofocusing Principles and Methods

中文版：离子交换色谱及色谱聚焦原理和方法

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！