Western blot

A Western blot, also called an immunoblot, picks out one particular protein from a complex mixture such as a cell extract or a blood sample and shows its approximate size and amount. The method was developed in 1979, and its name is a play on the Southern blot, an earlier technique for DNA.

Proteins are first denatured and separated by size with SDS-PAGE. An electric current then carries them out of the gel onto a membrane of nitrocellulose or PVDF, where they keep their positions. The membrane is blocked with milk protein or albumin so that antibodies cannot stick to it at random, then soaked in a primary antibody that binds only the target protein. A secondary antibody, which recognizes the first one and carries an enzyme or a fluorescent tag, makes light or color wherever the target sits. The result is a band whose position, judged against a ladder of marker proteins, reflects the protein's size.

Researchers use Western blots to check whether a gene is expressed, to compare protein levels between samples, usually against a loading control such as actin, and, with antibodies to phosphorylated forms, to follow cell signaling. In medicine they have served as confirmatory tests, for example for antibodies to the bacterium that causes Lyme disease.