Assessment of Cyclooxygenase Protein Expression by Western Blotting

Abstract

Western blotting provides an effective, sensitive, and specific method for the identification and quantitation of cyclooxygenase protein expression in an unknown complex protein sample. Electrophoresis is used to separate complex mixtures of proteins. Denaturing discontinuous one-dimensional gel electrophoresis separates proteins only based on molecular size as they move through a SDS-polyacrylamide gel (SDS-PAGE) toward the anode with the smaller proteins migrating faster and bigger proteins running slower. The SDS-PAGE is a separating gel topped by a stacking gel and secured in an electrophoresis apparatus. Sample proteins are solubilized by boiling in the presence of SDS and equal amounts of the protein solution are loaded into a gel lane, and the individual proteins separated electrophoretically. 2-Mercaptoethanol (2-ME) and dithiothreitol (DTT) are added to reduce disulfide bonds.