The western blot (sometimes called the protein immunoblot) is a widely used analytical technique used to detect specific proteins in a sample of tissue homogenate or extract.
Western- or immunoblotting is a commonly employed technique for the detection of protein antigens in complex mixtures. 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.
There are many reagent companies that specialize in providing antibodies (both monoclonal and polyclonal antibodies) against tens of thousands of different proteins. Commercial antibodies can be expensive, although the unbound antibody can be reused between experiments. This method is used in the fields of molecular biology, immunogenetics and other molecular biology disciplines.
It is not uncommon that, contrary to the theoretical predictions, several bands are detected. Although it is possible that an antibody is not entirely specific for the protein, other factors may be responsible.
Western Blot / WB Introduction
Western Blot / WB protocol
Western Blot / WB Tips
- Sinobiological Western blot general tips
- 1. General Tips to consider when performing a western blot
- 2. Sinobiological Western Blot Conditions
- 3. Why is the actual western blot band size different from the predicted?
- 4. Why do I see a weak signal or no signal at all on my western blot?
- 5. Will this antibody react with the same target in a different species?
- 6. Why are there extra bands on my western blot?
- 7. Do my transfer conditions change if my protein of interest if >180kD?
- Western Blotting: 10 Technical Tips for Success
- 1. Determine the best usage of the target protein and primary antibody
- 2. Keep up the protein transfer efficiency
- 3. Anticipate the effect of gel thickness in western blot
- 4. Make sure to equilibrate membranes and gels on transfer solution
- 5. Cleaner blots with the right blocking solution
- 6. Optimize your incubation time
- 7. Sometimes antibodies won't recognize denatured proteins
- 8. Western blot of phosphorylated proteins
- 9. Detection of low abundance proteins
- 10. Stripping and re-probing membranes
Western Blot / WB protocolWestern Blot / WB FAQ