Western Blot Guide

Western Blot Workflow

In standard Western blot (WB) analysis, proteins are first separated according to their molecular weight using denaturing SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis). The separated proteins are then transferred onto a membrane, where they are detected using specific antibodies.

SDS (sodium dodecyl sulfate) is an anionic detergent that denatures proteins and uniformly coats them with a negative charge. This treatment ensures that protein migration during electrophoresis depends primarily on size rather than intrinsic charge or shape. The addition of reducing agents such as β-mercaptoethanol or DTT (dithiothreitol) breaks disulfide bonds, and heating the samples promotes complete protein unfolding.

Proteins of interest are typically detected using a primary antibody that specifically binds the target protein. A secondary antibody, directed against the primary antibody, is then applied. This secondary antibody is conjugated to an enzyme or fluorophore, enabling visualization of the signal.