Immunoblotowanie

Immunoblotowanie, often referred to as Western blotting, is a common laboratory technique used for the detection and analysis of specific proteins in a complex mixture of proteins. This method combines gel electrophoresis, which separates proteins based on size, with immunological detection methods. The process begins with separating proteins from a biological sample, such as cell lysates or tissue extracts, using gel electrophoresis, typically SDS-PAGE. Following separation, the separated proteins are transferred from the gel onto a solid support membrane, most commonly nitrocellulose or PVDF. This transfer creates a replica of the protein separation pattern on the membrane.

The next crucial step involves blocking the membrane with a protein solution, like non-fat dry milk or bovine serum albumin, to prevent non-specific binding of antibodies. Subsequently, the membrane is incubated with a primary antibody that is specifically designed to bind to the target protein of interest. After washing away unbound primary antibody, a secondary antibody is added. This secondary antibody is conjugated to an enzyme or a fluorescent molecule and is designed to bind to the primary antibody. After another washing step, a substrate is added that reacts with the enzyme conjugate to produce a detectable signal, such as a colored precipitate or luminescence. This signal allows for the visualization and quantification of the target protein, indicating its presence, relative abundance, and molecular weight. Immunoblotowanie is a versatile tool used in various fields, including molecular biology, biochemistry, and medical diagnostics.