Immunoblot

An immunoblot, commonly called a Western blot, is a laboratory technique used to detect a specific protein within a complex sample. The method combines protein separation by gel electrophoresis with antibody-based detection. Proteins are first separated by size using SDS-PAGE and then transferred to a durable membrane such as nitrocellulose or PVDF. The membrane is blocked to reduce non-specific binding and incubated with a primary antibody that recognizes the target protein. After washing, a secondary antibody that binds to the primary antibody is applied; this secondary antibody is typically linked to an enzyme or fluorescent label. Visualization occurs through chemiluminescent, fluorescent, or colorimetric detection, producing bands corresponding to the target protein’s size as estimated by a molecular weight marker.

Applications and variants: Immunoblotting is widely used to confirm the presence of a protein, assess expression levels, or verify results from other assays. It can detect post-translational modifications with modification-specific antibodies and can be adapted for multiplexed detection. Stripping and reprobing the membrane allows sequential testing for multiple proteins. Controls such as loading controls (for example actin or tubulin) and positive/negative controls are important to interpret results.

Limitations and considerations: The technique is semi-quantitative and highly dependent on antibody quality and binding specificity. It can be time-consuming and requires careful optimization of blocking, antibody concentrations, and detection methods. Immunoblotting remains a standard tool in research and diagnostic settings for protein identification and verification.