Immunoblottaggi

Immunoblottaggi, or immunoblotting, is a laboratory technique used to detect a specific protein within a complex mixture. In English, the method is commonly called Western blotting. It combines gel electrophoresis for protein separation with antibody-based detection to identify the protein of interest and assess its size and relative abundance.

Principle and workflow: Proteins are extracted, separated by size using SDS-PAGE, and then transferred onto a membrane (nitrocellulose or PVDF) by electroblotting. After blocking to reduce non-specific binding, the membrane is probed with a primary antibody specific to the target protein, followed by a labeled secondary antibody. Detection relies on signals from the label, most often chemiluminescent or fluorescent, which are captured on film or by a digital imager. The observed signal correlates with the amount of target protein, generally after normalization to a loading control.

Applications and considerations: Immunoblottings are used to confirm protein identity, monitor expression changes across samples, verify post-translational modifications with modification-specific antibodies, and validate results from other methods. Rigorous controls are essential, including positive and negative controls and loading controls (e.g., actin, GAPDH) or total protein staining. Limitations include dependence on antibody quality and specificity, semi-quantitative interpretation, potential signal saturation, and loss of native protein conformation due to denaturation. Variants include dot blot and far-western blot, as well as immunoprecipitation followed by blotting to enrich low-abundance proteins.