Interpreting a Western Blot Test

A western blot test blot test is an important tool for the diagnosis of infectious diseases such as Lyme disease and HIV infection. However, it is often difficult to interpret the results of a western blot and there are other antibody/antigen diagnostic tests that give more accurate and faster results. A western blot can help to confirm the presence of a protein in a sample, but does not provide information about its exact location or structure.

In a western blot, proteins are separated on a polyacrylamide gel based on their molecular weight using an electric field. The proteins are denatured and loaded into individual wells of a gel with one lane reserved for a commercially available mixture of proteins of known molecular weights, called a ladder (figure below). When voltage is applied to the electrophoresis gel, the different sizes of proteins move at different rates through the gel (different electrophoretic mobilities), which separates them into bands. The band patterns can then be compared to the ladder bands, which allows estimation of the proteins’ molecular weights.

Western Blot Test: What It Is and When to Use It

The separating gel is transferred to a nitrocellulose or PVDF membrane that has been blocked with milk or other blocking agent to prevent nonspecific binding of antibodies. The membrane is then exposed to a primary antibody that binds to the target protein. This is followed by a secondary antibody that recognizes the Fc region of the first antibody and produces a colorimetric reaction that can be detected with a suitable chromogenic stain, fluorescent probe or radioactive label.