Western Blotting Handbook and Troubleshooting Guide

Table of Contents
Introduction1
Western Blotting Overview2-3
Transfer Protein to a Membrane4
Transfer Buffers4
Nitrocellulose Membrane 4
Polyvinylidene Difluoride Membrane 4
Filter Paper for Blotting4
MemCode™ Protein Stains5
Molecular Weight Markers 6-7
Miser™ Antibody Extender NC8
Increasing Sensitivity of a Western Blot9
Qentix™ Western Blot Signal Enhancer 9
Blocking Nonspecific Binding Sites 10
Transfer Membranes 10
Blocking Buffer Optimization 11
Blocking Buffers12-13
Washing the Membrane14
Wash Buffers 14
Primary and Secondary Antibodies 15
Conjugate Stabilizer Solutions 16
DyLight™ Fluor Antibody and Streptavidin Conjugates17
Affinity-Purified Secondary Antibodies 18-21
Labeling Your Own Antibodies22-25
ImmunoPure® Enzymes 23
EZ-Link™ Activated Enzymes 24-25
EZ-Label™ Fluorescent Labeling Kits26
DyLight™ Reactive Fluors27
Optimizing Antibody Concentration 28-29
Chromogenic Substrates30-31
Chemiluminescent Substrates32-39
Pierce ECL Substrate 33
SuperSignal® Chemiluminescent Substrates 34-38
LumiPhos™ Chemiluminescent Substrate 39
Quick Reference Substrate Guide 39
Data Imaging40
CL-XPosure™ Film 40
Western Blot Detection Kits41-42
Far-Western Blotting43-44
ProFound™ Far Western Protein:Protein Interaction Kits 45
In-Gel Western Detection 45-47
UnBlot® In-Gel Chemiluminescent Detection Kit47
Optimizing the Signal-to-Noise Ratio 48
Restore™ Western Blot Stripping Buffer50
Erase-It® Background Eliminator 51-52
Troubleshooting Guide – Blotting with Chemiluminescence53-57
Western Blotting Protocol Using Chemiluminescent Substrates 58-59
Recommended Reading60