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

今天我们就发布第二期：
Gel Filtration Principles and Methods
凝胶过滤原理和方法

英文版：Gel Filtration Principles and Methods

Contents
Introduction ............................................................................................................. 5
Symbols .....................................................................................................................................................6
Common abbreviations ...............................................................................................................................7
Chapter 1
Gel filtration in practice ............................................................................................ 9
Introduction ................................................................................................................. 9
Separation by gel filtration ............................................................................................. 9
Resolution in gel filtration ........................................................................................... 13
Media selection .......................................................................................................... 16
Sample preparation ..................................................................................................... 20
Buffer composition and preparation .............................................................................. 21
Denaturing (chaotropic) agents and detergents .............................................................................................22
Column and media preparation .................................................................................... 23
Sample application ..................................................................................................... 23
Elution and flow rates ................................................................................................. 24
Method development for high resolution fractionation .................................................... 26
Care of gel filtration media .......................................................................................... 27
Equipment selection ................................................................................................... 27
Scaling up ................................................................................................................. 27
BioProcess Media for large-scale production .................................................................. 29
Troubleshooting .......................................................................................................... 29
Chapter 2
Gel filtration media ................................................................................................. 35
Components of gel filtration media ............................................................................... 35
Superdex: first choice for high resolution, short run times and high recovery ..................... 36
Separation options ....................................................................................................................................38
Separation examples .................................................................................................................................39
Performing a separation ..............................................................................................................................42
Cleaning ..................................................................................................................................................43
Media characteristics ................................................................................................................................44
Chemical stability .....................................................................................................................................44
Storage ....................................................................................................................................................44
Sephacryl: fast, high recovery separations at laboratory and industrial scale ..................... 45
Separation options ....................................................................................................................................48
Separation examples .................................................................................................................................48
Performing a separation ..............................................................................................................................49
Cleaning ..................................................................................................................................................50
Media characteristics ................................................................................................................................51
Chemical stability .....................................................................................................................................51
Storage ....................................................................................................................................................51
Superose: broad fractionation range, but not suitable for industrial scale separations ........ 52
Separation options ....................................................................................................................................53
Separation examples .................................................................................................................................54
Performing a separation ..............................................................................................................................54
Cleaning ..................................................................................................................................................55
Media characteristics ................................................................................................................................56
Chemical stability .....................................................................................................................................56
Storage ....................................................................................................................................................56
Sephadex: rapid group separation of high and low molecular weight substances,
such as desalting, buffer exchange and sample clean up ................................................ 57
Separation options ....................................................................................................................................59
Separation examples .................................................................................................................................61
Performing a separation ..............................................................................................................................61
Scale up and processing large sample volumes .............................................................................................66
Media characteristics ................................................................................................................................68
Column Packing ........................................................................................................................................68
Cleaning ..................................................................................................................................................68
Chemical stability .....................................................................................................................................68
Storage ....................................................................................................................................................69
Chapter 3
Gel filtration in theory ............................................................................................. 71
Defining the process ................................................................................................... 71
Selectivity curves and media selection .......................................................................... 74
Resolution ................................................................................................................. 75
Chapter 4
Molecular weight determination and molecular weight distribution analysis .............. 79
Chapter 5
Sephadex LH-20 ..................................................................................................... 81
Separation options ....................................................................................................................................82
Separation examples .................................................................................................................................82
Packing a column .....................................................................................................................................83
Performing a separation ..............................................................................................................................84
Cleaning ..................................................................................................................................................84
Medium characteristics ..............................................................................................................................84
Chemical stability .....................................................................................................................................85
Storage ....................................................................................................................................................85
Transferring Sephadex LH-20 between organic solvents .................................................................................85
Chapter 6
Gel filtration in a Purification Strategy (CIPP) ............................................................ 87
Applying CIPP ............................................................................................................. 87
Selection and combination of purification techniques ..................................................... 88
Gel filtration as a polishing step ..................................................................................................................91
4
Appendix 1
Column packing and preparation ............................................................................. 93
Columns for packing gel filtration media ....................................................................... 93
Checking column efficiency ......................................................................................... 95
Column packing for high resolution fractionation using Superdex prep grade and
Sephacryl High Resolution ........................................................................................... 95
Column packing for group separations using Sephadex ................................................... 98
Appendix 2
Sephadex and Darcy’s law .................................................................................... 102
Appendix 3
Sample preparation .............................................................................................. 103
Sample stability ......................................................................................................................................103
Sample clarification ................................................................................................................................104
Specific sample preparation steps .............................................................................. 105
Removal of lipoproteins ............................................................................................. 108
Removal of phenol red .............................................................................................. 108
Removal of low molecular weight contaminants ........................................................... 108
Appendix 4
Selection of purification equipment ....................................................................... 109
Appendix 5
Converting from linear flow (cm/hour) to volumetric
flow rates (ml/min) and vice versa ......................................................................... 110
Appendix 6
Conversion data: proteins, column pressures ......................................................... 111
Appendix 7
Table of amino acids ........................................................................................... 112
Appendix 8
Analytical assays during purification ..................................................................... 114
Appendix 9
Storage of biological samples ............................................................................... 116
Additional reading and reference material ............................................................. 117
Ordering information ............................................................................................. 118

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中文版：凝胶过滤原理和方法

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！