Western Blot: Principles, Procedures, and Clinical Applications

Excerpt

The term "Western blot" was introduced by Burnette in 1981, following the development of the Southern blot for DNA and the Northern blot for RNA in 1977. Western blotting separates, detects, and identifies specific proteins within complex mixtures.

The technique entails separating proteins by polyacrylamide gel electrophoresis (PAGE), after which the resolved proteins are immobilized onto a nitrocellulose or nylon membrane using an electric current that drives their transfer. Protein detection on the membrane is achieved using antibodies labeled with probes, including radioactive isotopes or enzymes. The use of labeled probes enhances detection sensitivity, achieving limits 10- to 100-fold lower than those attainable by direct immunoprecipitation or protein staining methods. Densitometric analysis of band intensity allows quantitative comparison of protein expression across experimental conditions, such as treatment effects or temporal variations.

The Centers for Disease Control and Prevention no longer supports the use of the Western blot assay for diagnostic purposes. However, the technique remains integral to biomedical research for protein identification, quantification, and posttranslational modification analysis. Western blotting validates gene expression studies, confirms antibody specificity, and assesses signaling pathway activation in experimental models. In clinical laboratories, the assay supports confirmatory testing for infectious and autoimmune diseases, detection of disease-specific biomarkers, and evaluation of therapeutic protein expression in recombinant systems.