Western blot analysis is a useful tool for the detection of proteins in biological samples.  In order to obtain good results, high quality, specific antibodies (monoclonal or polyclonal) must be used.

It is important to realize that this technique is not ideal for determining the absolute expression level of a given protein (ion channel, enzyme, scaffolding protein or other peptide).

Here is a basic assay summary:

- Proteins are separated by gel electrophoresis

- Proteins are transferred to a membrane (ex. PVDF), spaced out by the electrophoresis

- The blot is incubated with non fat dry milk (NFDM) to bind to any remaining sites on the membrane. An antibody is then added to the solution to bind to its specific target protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it that serves to aid in visualization.

- The location of the antibody is revealed by incubating it with a substrate and exposing a film to the reaction

- The band of interest is seen by developing the film

- Band density is compared using specialized software and a good quality optical scanner.

Depending on the antobody application protocol one applies (i.e. overnight incubation or 1 hour antibody incubation with the membrane), the protocol takes 1 or 2 days to complete.