Western blot

The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract, and to visualize, distinguish, and quantify the different proteins in a complicated protein combination.

Western blot technique uses three elements to achieve its task of separating a specific protein from a complex: separation by size, transfer of protein to a solid support, and marking target protein using a primary and secondary antibody to visualize. A synthetic or animal-derived antibody (known as the primary antibody) is created that recognizes 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 recognizes and binds to the primary antibody. The secondary antibody is visualized 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, English biologist Edwin Southern. Similarly, detection of RNA is termed as northern blot. The term western blot was given by W. Neal Burnette in 1981, although the method, but not the name, was independently invented in 1979 by Jaime Renart, Jakob Reiser, and George Stark, and by Harry Towbin, Theophil Staehelin, and Julian Gordon at the Friedrich Miescher Institute in Basel, Switzerland. The Towbin group also used secondary antibodies for detection, thus resembling the actual method that is almost universally used today. Between 1979 and 2019 "it has been mentioned in the titles, abstracts, and keywords of more than 400,000 PubMed-listed publications" and may still be the most-used protein-analytical technique.

The western blot is extensively used in biochemistry for the qualitative detection of single proteins and protein-modifications (such as post-translational modifications). At least 8–9% of all protein-related publications are estimated to apply western blots. 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 colour 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.

Further applications of the western blot technique include its use by the World Anti-Doping Agency (WADA). Blood doping is the misuse of certain techniques and/or substances to increase one's red blood cell mass, which allows the body to transport more oxygen to muscles and therefore increase stamina and performance. There are three widely known substances or methods used for blood doping, namely, erythropoietin (EPO), synthetic oxygen carriers and blood transfusions. Each is prohibited under WADA's List of Prohibited Substances and Methods. The western blot technique was used during the 2014 FIFA World Cup in the anti-doping campaign for that event. In total, over 1000 samples were collected and analysed by Reichel, et al. in the WADA accredited Laboratory of Lausanne, Switzerland. Recent research utilizing the western blot technique showed an improved detection of EPO in blood and urine based on novel Velum SAR precast horizontal gels optimized for routine analysis. With the adoption of the horizontal SAR-PAGE in combination with the precast film-supported Velum SAR gels the discriminatory capacity of micro-dose application of rEPO was significantly enhanced.

For medication development, the identification of therapeutic targets, and biological research, it is essential to comprehend where proteins are located within a cell. The subcellular locations of proteins inside the cell and their functions are closely related. The relationship between protein function and localization suggests that when proteins move, their functions may change or acquire new characteristics. A protein's subcellular placement can be determined using a variety of methods. Numerous efficient and reliable computational tools and strategies have been created and used to identify protein subcellular localization. With the aid of subcellular fractionation methods, WB continues to be an important fundamental method for the investigation and comprehension of protein localization.