What are the principles of western blotting?

Western blot samples are initially prepared by extracting proteins using specialized cell lysis buffers containing protease and phosphatase inhibitors. It's essential to ensure that each sample contains an equal concentration of proteins for accurate analysis. Proteins are then separated based on their molecular weight using techniques such as SDS-PAGE. Next, the separated proteins are transferred from the gel onto a solid support membrane (blotting), typically made of nitrocellulose or polyvinylidene difluoride (PVDF), to allow further analysis. Multiple blotting methods are available, but the fundamental principle of electrophoretic transfer remains consistent. In blotting, a transfer sandwich with a modified electrode buffer is utilized. The target protein of interest is then identified using specific primary antibodies that bind to the target protein. These antibodies are then detected using secondary antibodies conjugated to enzymes or fluorophores, allowing visualization of the target protein bands.