Overview
In this protocol, samples containing the target protein are deposited onto a polyvinyldifluoride (PVDF) membrane using a vacuum manifold. The immobilized protein is exposed to an antibody specific for the target protein, followed by an antibody that reacts with species-specific determinants carried by the primary antibody and is conjugated to horseradish peroxidase (HRP). Antibody complexes captured by the immobilized target protein are detected by enhanced chemiluminescence (ECL); in the presence of H2O2, HRP converts luminol to an excited intermediate dianion that emits blue light (428 nm) on return to ground state (Isacsson and Wettermark 1974).
Details
Details of this protocol, Measuring Protein Concentration By Western Analysis Using Enhanced Chemiluminescence Detection-SUBSCRIPTION REQUIRED, are located on a web site other than Biocompare Protocols.