Western blotting

Abstract

Western blotting is employed for the detection of proteins and other macromolecules immobilized on nitrocellulose membranes. This rapid and sensitive method enables the identification and quantification of a specific protein from cell lysates or a mixture of proteins. Following the resolution on denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE), the constituent polypeptides are transferred electrophoretically to a nitrocellulose membrane (1). The membrane is incubated in a solution containing primary antibody and the resultant antigen-antibody complex is detected by using appropriately labeled ligands. The most common method is based on the enzyme-linked immunodetection of antigen-specific antibodies using anti-IgG secondary antibodies conjugated with either horseradish peroxidase (HRP) or alkaline phosphatase (AP). Visualization of antibodyantigen complex is achieved through the use of an enhanced chemiluminiscent (ECL) method. It is possible to detect as little as 10-50 ng of protein with AP-conjugated secondary antibody and 0.5-1.0 ng of protein with HRP-conjugated secondary antibody using high affinity and high titer primary antibodies.