Western Blotting (also known as Protein Immunoblotting) is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract. Samples are first separated by SDS-polyacrylamide gel electrophoresis. The separated proteins are then transferred to a membrane (usually nitrocellulose, polyvinyl pyrolidon, or nylon). These membranes are incubated with an antibody specific for the protein of interest which binds to the protein band immobilized on the membrane. The antibody is then visualized with a detection system that is usually based on a secondary protein binding to Ig chains which is linked to a color-yielding reaction.
|Preparation for Western Blot||→||Electrophoresis & Transfer & Blocking||→||Antibody incubation||→||Detection & Analysis|
|• WB Buffers / Reagents Recipes
• Sample Preparation
|• Gel Electrophoresis
• Protein Transfer Methods
• Membrane Blocking
|• Primary Antibody Incubation
• Secondary Antibody Incubation
|• WB Detection
• Densitometric Analysis
The following Western Blotting protocols are for your convenience to perform a Western Blotting test. No single protocol is ideal for every Western Blotting experiment since experiment conditions change. Empirical testing of a Western Blotting protocol is essential.
Although Western Blotting is a common technique to detect specific proteins, researchers often feel frustrated with unsatisfactory results of Western Blotting. The following list 10 problems that Western Blotters may have encountered.
Frequently asked questions about Western Blotting: Western Blotting FAQ.