Western blotting

Western blotting is a widely used analytical technique in molecular biology and genetics to detect specific proteins in a sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein.

History

The technique of Western blotting was developed in the late 1970s by scientists at the University of California, San Francisco, as a method of identifying specific proteins in complex mixtures. The name "Western blot" was given to the technique as a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. The name is also a pun on the Northern blot technique which was developed for RNA detection. Thus, Western blotting was the third major technique of this type to be developed.

Procedure

The process begins with the preparation of a protein sample, which is then loaded into a gel. The gel is subjected to an electric current, causing the proteins to migrate through the gel. The proteins are then transferred to a membrane, which is then blocked to prevent non-specific binding of the antibodies. The membrane is then incubated with a primary antibody specific to the target protein, followed by a secondary antibody that is usually linked to an enzyme. The enzyme catalyzes a reaction that produces a visible signal, allowing for the detection of the target protein.

Applications

Western blotting has a wide range of applications, including the diagnosis of diseases such as HIV and Lyme disease, the analysis of protein expression in cell cultures, and the determination of protein size and abundance. It is also used in research to study the effects of drugs and other treatments on protein expression.

Limitations

While Western blotting is a powerful tool, it does have some limitations. These include the need for high-quality antibodies, the potential for non-specific binding, and the inability to determine the exact location of a protein within a cell.

See also

• Southern blot
• Northern blot
• Dot blot
• Immunohistochemistry
• ELISA