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：
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

英文版：Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods

Contents
Introduction ........................................................................................................................................ 7
Symbols ................................................................................................................................................................................................. 8
Common abbreviations .................................................................................................................................................................. 8
Chapter 1
Principles of hydrophobic interaction chromatography................................................. 9
Hydrophobic interaction chromatography in theory ..............................................................................10
The role of water ............................................................................................................................................................................ 10
Protein structure ............................................................................................................................................................................. 10
Reversible interactions ................................................................................................................................................................ 11
Steps in a HIC separation ........................................................................................................................................................... 11
Resolution .......................................................................................................................................................................................... 14
Efficiency ........................................................................................................................................................................................... 15
Selectivity ........................................................................................................................................................................................... 16
Selectivity and binding capacity ......................................................................................................................................................................... 16
Selectivity and salt selection ................................................................................................................................................................................. 16
Selectivity and the properties of a HIC medium ........................................................................................................................................... 17
Selectivity and elution .............................................................................................................................................................................................. 20
Chapter 2
Hydrophobic interaction chromatography in practice ................................................ 23
Introduction ................................................................................................................................................................23
HIC in a purification strategy (Cipp) ....................................................................................................................................... 23
Capture ............................................................................................................................................................................................................................ 24
Intermediate purification ......................................................................................................................................................................................... 24
Polishing .......................................................................................................................................................................................................................... 24
Practical considerations for HIC separation .................................................................................................25
Screening for selectivity ........................................................................................................................................28
Automated HIC media screening, method development and optimization ........................................................ 28
Manual media screening, method development and optimization ......................................................................... 29
Screening for selectivity using HiTrap HIC Selection Kit ............................................................................................................................ 30
Screening for binding (salt) conditions .............................................................................................................................................................. 30
Optimization of gradient, flow rate and sample loading ........................................................................................................................... 30
Sample properties and choice of ligand ............................................................................................................................... 31
Salt selection and buffer preparation ................................................................................................................................... 32
Salts ................................................................................................................................................................................................................................... 32
Salt concentration ...................................................................................................................................................................................................... 33
Buffer ions and pH ...................................................................................................................................................................................................... 34
Buffer additives ............................................................................................................................................................................................................ 35
Column and media preparation .............................................................................................................................................. 36
Sample preparation ...................................................................................................................................................................... 36
Concentration and viscosity .................................................................................................................................................................................. 37
Sample application ........................................................................................................................................................................ 37
Sample load ...................................................................................................................................................................................... 38
Sample volume ............................................................................................................................................................................... 38
Temperature ..................................................................................................................................................................................... 38
Elution ............................................................................................................................................................................39
Linear gradient elution ................................................................................................................................................................ 39
Handbook 11-0012-69 AA 3
Step elution ....................................................................................................................................................................................... 41
Flow rates .......................................................................................................................................................................................... 42
Flow control ...................................................................................................................................................................................... 43
Wash and re-equilibration ...................................................................................................................................43
Analysis of results and further steps ................................................................................................................44
Scaling up ....................................................................................................................................................................44
Equipment selection ...............................................................................................................................................45
Care of HIC media ....................................................................................................................................................45
Troubleshooting ........................................................................................................................................................46
The ideal HIC separation: target protein is well resolved by gradient elution ................................................................................. 46
Target protein is eluted early in the gradient. Poor resolution. .............................................................................................................. 46
Target protein is eluted near the end of the gradient. Poor resolution. .............................................................................................. 46
Target protein is eluted in the middle of the gradient. Poor resolution. ............................................................................................. 46
BioProcess Media - made for bioprocessing ................................................................................................50
Custom Designed Media ............................................................................................................................................................. 50
Custom Products ............................................................................................................................................................................ 51
Chapter 3
Media for hydrophobic interaction chromatography ................................................... 53
Introduction ................................................................................................................................................................53
SOURCE: purification with high resolution and easy scale-up .............................................................53
Purification options ....................................................................................................................................................................... 54
Purification examples ................................................................................................................................................................... 56
Method optimization ................................................................................................................................................................................................. 56
Scaling up ....................................................................................................................................................................................................................... 57
Polishing .......................................................................................................................................................................................................................... 58
Performing a separation ............................................................................................................................................................. 59
First-time use or after long-term storage ............................................................................................................................ 59
Separation by gradient elution ................................................................................................................................................ 59
Separation by step elution ........................................................................................................................................................ 60
Cleaning ............................................................................................................................................................................................. 60
Media characteristics ................................................................................................................................................................... 61
Chemical stability .......................................................................................................................................................................... 61
Storage ............................................................................................................................................................................................... 61
Sepharose High Performance: purification with high resolution ........................................................62
Purification options ....................................................................................................................................................................... 62
Purification examples ................................................................................................................................................................... 64
Media screening: development of a monoclonal antibody purification ............................................................................................. 64
Capture: monoclonal antibody purification ................................................................................................................................................... 65
Intermediate purification: recombinant HIV reverse transcriptase ...................................................................................................... 65
Performing a separation ............................................................................................................................................................. 66
First-time use or after long-term storage ............................................................................................................................ 66
Separation by gradient elution ................................................................................................................................................ 66
Separation by step elution ........................................................................................................................................................ 67
Cleaning ............................................................................................................................................................................................. 68
Media characteristics ................................................................................................................................................................... 68
Chemical stability .......................................................................................................................................................................... 69
Storage ............................................................................................................................................................................................... 69
4 Handbook 11-0012-69 AA
Sepharose Fast Flow: purification with good resolution and easy scale-up .................................69
Purification options ....................................................................................................................................................................... 70
Purification examples ................................................................................................................................................................... 72
Media screening .......................................................................................................................................................................................................... 72
Capture: enzyme purification ................................................................................................................................................................................ 72
Capture: recombinant Hepatitis B virus surface antigen (r-HbsAg) from CHO cells ..................................................................... 73
Intermediate purification: Fab fragment .......................................................................................................................................................... 74
Intermediate purification: recombinant protein Annexin V ...................................................................................................................... 75
Performing a separation ............................................................................................................................................................. 75
First-time use or after long-term storage ............................................................................................................................ 76
Separation by gradient elution ................................................................................................................................................ 76
Separation by step elution ........................................................................................................................................................ 76
Cleaning ............................................................................................................................................................................................. 77
Media characteristics ................................................................................................................................................................... 78
Chemical stability .......................................................................................................................................................................... 78
Storage ............................................................................................................................................................................................... 78
Chapter 4
HIC in a purification strategy (Cipp) ....................................................................................... 79
Applying Cipp ................................................................................................................................................................................... 80
Selection and combination of purification techniques ............................................................................ 80
HIC as a capture step .............................................................................................................................................83
Purification of recombinant human epidermal growth factor (h-EGF) ................................................................... 83
HIC for intermediate purification .......................................................................................................................84
Purification of Fab fragment ..................................................................................................................................................... 85
HIC as a polishing step ..........................................................................................................................................86
Purification of a recombinant Pseudomonas aeruginosa exotoxin A, PE553D .................................................. 86
Alternative techniques for polishing steps .......................................................................................................................... 88
Chapter 5
Reversed phase chromatography: principles and methods ...................................... 89
Introduction ................................................................................................................................................................89
Terminology ...................................................................................................................................................................................... 90
RPC in theory .............................................................................................................................................................90
Steps in an RPC separation ....................................................................................................................................................... 91
Resolution .......................................................................................................................................................................................... 93
Efficiency ........................................................................................................................................................................................................................ 94
Selectivity ........................................................................................................................................................................................... 95
Components of an RPC medium .......................................................................................................................................................................... 95
Eluents ............................................................................................................................................................................................................................. 97
Ion-pairing agents ...................................................................................................................................................................................................... 99
Mixed-mode retention and ion suppression ................................................................................................................................................. 100
Elution ............................................................................................................................................................................................................................ 101
Binding capacity ....................................................................................................................................................................................................... 103
RPC in practice ....................................................................................................................................................... 103
Media and column selection ................................................................................................................................................... 104
Sample components: hydrophobicity ............................................................................................................................................................. 104
Handbook 11-0012-69 AA 5
Goal of separation .................................................................................................................................................................................................... 104
Scale of separation .................................................................................................................................................................................................. 105
Eluent conditions ....................................................................................................................................................................................................... 105
Column length ............................................................................................................................................................................................................ 105
Eluent selection and preparation ......................................................................................................................................... 105
pH and ion-pairing agents .................................................................................................................................................................................... 106
Organic modifiers ..................................................................................................................................................................................................... 106
Typical eluent protocols for separation of proteins and peptides ...................................................................................................... 106
Preparation .................................................................................................................................................................................................................. 107
Column and media preparation ............................................................................................................................................ 108
Sample preparation .................................................................................................................................................................... 108
Sample solubility ....................................................................................................................................................................................................... 108
Concentration and viscosity ................................................................................................................................................................................ 109
Sample load .................................................................................................................................................................................... 109
Sample volume ............................................................................................................................................................................. 109
Temperature ................................................................................................................................................................................... 109
Optimization ................................................................................................................................................................................................................ 110
Wash and re-equilibration ....................................................................................................................................................... 111
Troubleshooting ..................................................................................................................................................... 112
Ghosting ........................................................................................................................................................................................... 112
Baseline drift: balancing eluents ........................................................................................................................................... 112
μRPC C2/C18: for high-resolution separation of complex samples ................................................ 116
Purification options ..................................................................................................................................................................... 116
Separation examples ................................................................................................................................................................. 117
Performing a separation ........................................................................................................................................................... 118
First-time use or after long-term storage .......................................................................................................................... 118
Separation by gradient elution .............................................................................................................................................. 118
Cleaning ........................................................................................................................................................................................... 119
Media characteristics ................................................................................................................................................................. 120
Chemical stability ........................................................................................................................................................................ 120
Storage ............................................................................................................................................................................................. 120
SOURCE: rapid separation with high resolution and easy scale-up ............................................... 121
Purification options ..................................................................................................................................................................... 122
Purification examples ................................................................................................................................................................. 123
Capture and purification of a synthetic peptide ........................................................................................................................................ 123
Scaling up ..................................................................................................................................................................................................................... 124
Performing a separation ........................................................................................................................................................... 125
First-time use or after long-term storage .......................................................................................................................... 125
Separation by gradient elution .............................................................................................................................................. 125
Cleaning ........................................................................................................................................................................................... 126
Media characteristics ................................................................................................................................................................. 127
Chemical stability ........................................................................................................................................................................ 127
Storage ............................................................................................................................................................................................. 127
Reversed Phase Chromatography and Cipp ............................................................................................. 128
RPC as a capture step ................................................................................................................................................................ 128
RPC for intermediate purification ......................................................................................................................................... 128
RPC as a polishing step ............................................................................................................................................................. 128
6 Handbook 11-0012-69 AA
Appendix 1
Sample preparation ....................................................................................................................131
Sample stability ............................................................................................................................................................................ 131
Sample clarification .................................................................................................................................................................... 132
Specific sample preparation steps ................................................................................................................ 133
Resolubilization of protein precipitates ............................................................................................................................... 135
Buffer exchange and desalting ....................................................................................................................... 136
Removal of lipoproteins ..................................................................................................................................... 139
Removal of phenol red........................................................................................................................................ 139
Removal of low molecular weight contaminants ................................................................................... 139
Appendix 2
Column packing and preparation .........................................................................................141
Column selection .......................................................................................................................................................................... 143
Column packing and efficiency ............................................................................................................................................. 143
Appendix 3
Selection of purification equipment.....................................................................................145
Converting from linear flow (cm/hour) to volumetric flow rates (ml/min) and vice versa ........................... 146
From linear flow (cm/hour) to volumetric flow rate (ml/min) ................................................................................................................. 146
From volumetric flow rate (ml/min) to linear flow (cm/hour) ................................................................................................................. 146
From ml/min to using a syringe ......................................................................................................................................................................... 146
Appendix 4
Conversion data: proteins, column pressures .................................................................147
Column pressures ........................................................................................................................................................................ 147
Appendix 5
Table of amino acids...................................................................................................................148
Appendix 6
Analytical assays during purification .................................................................................151
Appendix 7
Storage of biological samples ................................................................................................153
Appendix 8
Column cleaning for HIC media.............................................................................................155
Removal of common contaminants .................................................................................................................................... 155
To remove lipids, lipoproteins and very hydrophobic proteins ................................................................................ 155
Product index ................................................................................................................................157
Additional reading........................................................................................................................158
References......................................................................................................................................159
Ordering information ..................................................................................................................160
Hydrophobic interaction media ...................................................................................................................... 160
Reversed phase media ....................................................................................................................................... 161
Other columns and accessories ...................................................................................................................... 162

更多内容请点击下载：Hydrophobic Interaction and Reversed Phase Chromatography Principles and Methods

中文版：疏水相互作用和反向层析技术原理和方法

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！