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Western Blotting Handbook and Troubleshooting Guide

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western blot principle and method guide
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 
Western Blotting Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3 
Transfer Protein to a Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 
MemCode™ Protein Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-5 
Blocking Nonspecific Binding Sites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 
Transfer Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 
Transfer Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 
Blocking Buffer Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 
Blocking Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-9 
Washing the Membrane. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 
Wash Buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 
Primary and Secondary Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 
Conjugate Stabilizer Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 
Affinity-Purified Secondary Antibodies . . . . . . . . . . . . . . . . . . . . . . . . 13-15 
Labeling Your Own Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 
ImmunoPure Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 
EZ-Link Activated Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18-19 
EZ-Label Fluorescent Labeling Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 
Optimizing Antibody Concentration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21-22 
Chromogenic Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23-24 
Chemiluminescent Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25-31 
SuperSignal Chemiluminescent Substrates . . . . . . . . . . . . . . . . . . . . 26-30 
LumiPhos Chemiluminescent Substrate . . . . . . . . . . . . . . . . . . . . . . . . 31 
Quick Reference Substrate Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 
Data Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 
CL-XPosure Film. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 
Western Blot Detection Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 
Far-Western Blotting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35-36 
In-Gel Western Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37-39 
Optimizing the Signal-to-Noise Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 
Restore Western Blot Stripping Buffer . . . . . . . . . . . . . . . . . . . . . . . . . 42 
Qentix Western Blot Signal Enhancer. . . . . . . . . . . . . . . . . . . . . . . . . . 43 
Erase-It Background Eliminator . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44-45 
Troubleshooting Guide – Blotting with Chemiluminescence . . . . . . . . . . . . . 46-49 
Western Blotting Protocol Using Chemiluminescent Substrates . . . . . . . . . . 50-51


Western Blotting Principles and Methods from GE Healthcare
 
Contents

Chapter 1 - Introduction....................................................... 9
Chapter 2 - Sample preparation.......................................15
2.1 Introduction................ 15
2.2 Protein extraction options..................................................... 16
2.2.1 Detergent-based lysis ............................................ 16
2.2.2 Freeze/thaw lysis...................................................... 16
2.2.3 Osmotic shock ........................................................... 17
2.2.4 Ultrasonication.......................................................... 17
2.2.5 Mechanical methods .............................................. 17
2.2.6 Enzymatic digestion................................................ 17
2.2.7 Explosive decompression .................................... 18
2.2.8 Pre-made lysis buffers and sample preparation kits ................................................ 18
2.2.9 Protecting your samples ....................................... 19
2.3 Sample cleanup........ 20
2.3.1 Sample cleanup products .................................... 22
2.3.2 Depletion of high abundance protein from serum or plasma samples ........... 23
2.3.3 Desalting and concentrating samples ........... 23
2.4 Determination of total protein concentration.............. 24
2.4.1 Products for determination of total protein concentration ................................... 25
2.5 References.................. 26
Chapter 3 - Gel electrophoresis ........................................27
3.1 Electrophoresis ......... 27
3.1.1 Polyacrylamide gels ................................................ 28
3.1.2 Buffer systems and pH .......................................... 31
3.1.3 Denaturing gels: SDS-PAGE ................................. 32
3.1.4 Native gels: PAGE...................................................... 33
3.1.5 2-dimensional (2-D) gel electrophoresis........ 33
3.1.6 Electrophoresis equipment from GE Healthcare......................................................... 34
3.2 Molecular weight markers .......................................36
3.2.1 Rainbow Molecular Weight Markers ............... 37
3.2.2 Amersham ECL DualVue Western Blotting Markers .................................................. 37
3.2.3 Amersham ECL Plex Fluorescent Rainbow Markers .................................................. 38
3.2.4 Unlabeled Protein Molecular Weight Markers.............................................................. 39
3.3 Determining the Mr of unknown proteins from molecular weight markers..................... 39
3.4 Total protein stains . 39
3.5 General polyacrylamide gel electrophoresis (PAGE) protocol................................................. 41
3.6 References.................. 42
Chapter 4 - Transfer ...........................................................43
4.1 Protein transfer......... 43
4.1.1 Electrotransfer........................................................... 43
4.1.2 Diffusion transfer...................................................... 50
4.2 Transfer buffers and running conditions........................ 51
4.2.1 Notes on transfer of large and small proteins.............................................................. 52
4.2.2 Current and transfer time ................................... 53
4.2.3 Monitoring and optimizing novel blotting protocols.................................................. 53
4.3 Membranes ................ 54
4.3.1 Nitrocellulose membranes from GE Healthcare.......................................................... 56
4.3.2 PVDF membranes from GE Healthcare ......... 56
4.3.3 Nylon-based membranes from GE Healthcare............................................................ 56
4.3.4 Membrane selection guide .................................. 57
4.4 Confirmation of protein transfer to the membrane.. 58
4.4.1 Total protein stains .................................................. 58
4.5 References.................. 60
Chapter 5 - Antibody probing............................................61
5.1 Blocking........................ 62
5.1.1 Proteins as blocking agents ................................ 62
5.1.2 Detergents as blocking agents.......................... 63
5.1.3 Buffers for blocking agents ................................. 64
5.1.4 Timing and temperature....................................... 64
5.2 Primary and secondary antibody probing..................... 64
5.2.1 Primary antibodies................................................... 64
5.2.2 Secondary antibodies............................................. 67
5.3 Stripping and reprobing membranes............................... 70
5.3.1 Stripping using heat and detergent................. 70
5.3.2 A strategy to optimize membrane stripping 70
5.3.3 Stripping using low pH........................................... 71
5.3.4 Stripping using high pH......................................... 71
5.3.5 Stripping using high salt solution...................... 72
5.3.6 Hints and tips ............................................................. 72
5.4 Automation................. 72
5.5 Blotting protocol ...... 73
5.6 References.................. 74
Chapter 6 - Detection .........................................................75
6.1 Chemiluminescence................................................................. 76
6.1.1 Amersham ECL .......................................................... 77
6.1.2 Amersham ECL Prime............................................. 78
6.1.3 Chemiluminescence hints and tips.................. 80
6.2 Fluorescence.............. 81
6.2.1 The multiplexing potential of Amersham ECL Plex..................................................... 83
6.2.2 Fluorescence hints and tips................................. 85
6.3 Chemifluorescence. 86
6.3.1 Chemifluorescence hints and tips .................... 88
Chapter 7 - Imaging ...........................................................89
7.1 Digital imaging.......... 89
7.1.1 CCD camera-based imagers............................... 89
7.1.2 Scanner systems ...................................................... 91
7.1.3 Fluorophores and filters ........................................ 92
7.2 Chemiluminescence detection on film ........................... 93
7.2.1 Flatbed scanners...................................................... 94
7.3 Autoradiography...... 94
7.3.1 X-ray film autoradiography................................. 94
7.3.2 Storage phosphor screen autoradiography 94
7.4 Detection system and imager compatibility ................ 94
Chapter 8 - Analysis ...........................................................95
8.1 Quantitative Western blotting............................................. 96
8.1.1 Sensitivity ... 96
8.1.2 Linear dynamic range............................................ 96
8.1.3 Signal stability............................................................ 97
8.1.4 In-lane normalization ............................................ 97
8.1.5 Signal-to-noise ratio ............................................... 99
8.2 Analysis software...101
8.2.1 Main features of ImageQuant TL software .................................................................101
Chapter 9 - Application examples ..................................105
9.1 Chemiluminescent Western blotting..............................106
9.1.1 Purification of recombinant proteins ............106
9.1.2 Analysis of IgG fractions purified from human plasma..........................................108
9.1.3 Detection of low abundance proteins:
Monitoring signaling pathway activation...110
9.1.4 Detection of protein interactions by co-immunoprecipitation
and Western blotting ...........................................112
9.2 Fluorescent Western blotting ............................................114
9.2.1 Multiplexed detection for normalizing against a housekeeping protein ......114
9.2.2 Multiplexed Western blotting and Deep Purple total protein staining ..........116
9.2.3 Multiplexed detection of proteins with similar molecular weights ...................118
9.2.4 Triplexed detection: The simultaneous detection of three proteins.................120
9.2.5 Three-layer fluorescent Western blotting for signal amplification...................122
9.2.6 2-D Western blotting for detection of phosphorylated isoforms ......................124
9.3 Reference ..................126
Chapter 10 - Troubleshooting ........................................127
10.1 Less than perfect results: A rogue's gallery ................137
10.2 Problems associated with running buffer and membrane handling................................138
10.3 Don't forget the simple things!..........................................139
10.4 Reference ..................140
Chapter 11 - Protocols and recipes ...............................141
11.1 Western blotting standard procedure...........................141
11.2 Sample preparation................................................................142
11.2.1 Extraction of proteins with Mammalian Protein Extraction Buffer ..................142
11.2.2 Sample clean up .....................................................144
11.2.3 Concentration measurement using the 2-D Quant Kit .........................................148
11.2.4 2× sample loading buffer ..................................149
11.3 Electrophoresis .......150
11.3.1 Preparation of polyacrylamide gels containing SDS ...............................................150
11.3.2 Electrophoresis running buffer for SDS-PAGE.............................................................152
11.4 Western blotting ....152
11.4.1 Wet transfer..............................................................152
11.4.2 Semidry transfer.....................................................154
11.5 Total protein stains .................................................................156
11.5.1 Deep Purple...............................................................156
11.5.2 Amersham AuroDye forte...................................157
11.6 Western blotting buffers ......................................................158
11.7 Blocking......................159
11.8 Antibody probing and detection.......................................159
11.8.1 Chemiluminescence detection with Amersham ECL
and Amersham ECL Prime ................................159
11.8.2 Chemifluorescence detection with Amersham ECF.................................................161
11.8.3 Fluorescence detection with Amersham ECL Plex....................................................161
11.8.4 Protocol for three layer probing ......................162
11.8.5 Conjugation of CyDye to antibody .................163
11.8.6 Affinity purification of antibodies....................164
11.9 Stripping and reprobing .......................................................166
11.9.1 Stripping using high pH and high temperature .........................................................167
11.9.2 Stripping using low pH.........................................167
11.9.3 Stripping using high pH.......................................167
11.9.4 Stripping using high salt solution....................167
11.10 Optimization protocols for finding optimal primary
and secondary antibody concentrations.....................168
11.10.1 Optimization of primary antibody concentration .....................................................169
11.10.2 Optimization of secondary antibody concentration................................................169
11.11 Reference ..................169
Chapter 12 - Glossary ......................................................171
Appendix A - Optimization ...............................................177
A.1 Choice of membrane............................................177
A.2 Choice of blocking agent....................................178
A.3 Choice of washing buffer....................................178
A.4 Optimization of antibody concentrations using membrane strips...................178
 
 
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