Western Blot? Wot that?

A western blot (a.k.a immunoblot) is a method in molecular biology/biochemistry/immunogenetics to detect protein in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate denatured proteins by mass. The proteins are then transferred out of the gel and onto a membrane (typically nitrocellulose), where they are "probed" using antibodies specific to the protein. As a result, researchers can examine the amount of protein in a given sample and compare levels between several groups. Other techniques also using antibodies allow detection of proteins in tissues (immunohistochemistry) and cells (immunocytochemistry).

The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette (Analytical Biochemistry, 112:195-203, 1981) and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern (Journal of Molecular Biology 98 (3): 503-&1975 ) [ See http://garfield.library.upenn.edu/histcomp/southern-em_auth/ for History of Citation ]. Detection of RNA is termed northern blotting.

Western blots allow investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples.

1) Proteins are separated by gel electrophoresis, usually SDS-PAGE.

2) The proteins are transfered to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel.

3) The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or dye attached to it which cannot be seen at this time.

4) The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed.