Western blot

The western blot (sometimes called the protein immunoblot) is a widely used analytical technique in molecular biology, immunogenetics and other molecular biology disciplines to detect specific proteins in a sample of tissue homogenate or extract. The western blot (sometimes called the protein immunoblot) is a widely used analytical technique in molecular biology, immunogenetics and other molecular biology disciplines to detect specific proteins in a sample of tissue homogenate or extract. In brief, the sample undergoes protein denaturation, followed by gel electrophoresis. A synthetic or animal-derived antibody (known as the primary antibody) is created that recognises and binds to a specific target protein. The electrophoresis membrane is washed in a solution containing the primary antibody, before excess antibody is washed off. A secondary antibody is added which recognises and binds to the primary antibody. The secondary antibody is visualised through various methods such as staining, immunofluorescence, and radioactivity, allowing indirect detection of the specific target protein. Other related techniques include dot blot analysis, quantitative dot blot, immunohistochemistry and immunocytochemistry, where antibodies are used to detect proteins in tissues and cells by immunostaining, and enzyme-linked immunosorbent assay (ELISA). The name western blot is a play on the Southern blot, a technique for DNA detection named after its inventor, British biologist Edwin Southern. Similarly, detection of RNA instead of proteins or DNA is termed as northern blot. The term 'western blot' was given by W. Neal Burnette, although the method itself originated in the laboratory of Harry Towbin at the Friedrich Miescher Institute in Basel, Switzerland. The western blot is extensively used in biochemistry for the qualitative detection of single proteins and protein-modifications (such as post-translational modifications). It is used as a general method to identify the presence of a specific single protein within a complex mixture of proteins. A semi-quantitative estimation of a protein can be derived from the size and color intensity of a protein band on the blot membrane. In addition, applying a dilution series of a purified protein of known concentrations can be used to allow a more precise estimate of protein concentration. The western blot is routinely used for verification of protein production after cloning. It is also used in medical diagnostics, e.g., in the HIV test or BSE-Test. The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as above. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary anti-human antibody linked to an enzyme signal is added. The stained bands then indicate the proteins to which the patient's serum contains antibody. A western blot is also used as the definitive test for variant Creutzfeldt-Jakob Disease, a type of prion disease linked to the consumption of contaminated beef from cattle with Bovine spongiform encephalopathy (BSE, commonly referred to as 'mad cow disease'). Another application is in the diagnosis of tularemia. An evaluation of the western blot’s ability to detect antibodies against F. tularensis revealed that its sensitivity is almost 100% and the specificity is 99.6%. Some forms of Lyme disease testing employ western blotting. A western blot can also be used as a confirmatory test for Hepatitis B infection and HSV-2 (Herpes Type 2) infection. In veterinary medicine, a western blot is sometimes used to confirm FIV+ status in cats.