Western blotting is an immunoassay technique used to detect specific proteins in a complex mixture. It combines protein separation by size and specific antibody binding to identify and semi‑quantify the presence of target antigens.
Principle and procedure
In a Western blot, proteins from a cell lysate or purified sample are first denatured and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‑PAGE). The separated proteins are then transferred electrophoretically onto a nitrocellulose or polyvinylidene difluoride membrane, preserving their relative positions. The membrane is blocked with a protein solution such as bovine serum albumin to prevent nonspecific binding, then incubated with a primary antibody that recognizes the protein of interest. After washing, a secondary antibody conjugated to an enzyme or fluorescent probe binds to the primary antibody. Upon addition of substrate, the enzyme catalyses a reaction that produces a chemiluminescent, chromogenic or fluorescent signal at the site of the target protein band. The intensity of the signal correlates with the amount of antigen.
Applications and considerations
Western blots are widely used in biomedical research to confirm the size and expression level of proteins, to verify the specificity of antibodies and to detect post‑translational modifications. Clinically, immunoblotting has served as a confirmatory test for infection with HIV‑1/2, Lyme disease and certain autoimmune disorders, though newer assays have replaced or supplemented it in some settings. The technique can detect prion proteins in Creutzfeldt–Jakob disease and has been used to screen for viral proteins in Zika and Ebola research. Limitations include the need for high-quality antibodies, time‑consuming procedures and potential for cross-reactivity or false positives. Semi‑quantitative interpretation requires normalization to a housekeeping protein and careful control of loading and transfer conditions.
Western blotting remains a versatile tool for assessing protein expression and integrity, bridging electrophoretic separation with antibody specificity.
Related Terms: SDS-PAGE, Antibody, Immunoassay, Protein electrophoresis, ELISA