Western Immunoblot Analysis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( 1 ) combined with Western immunoblot analysis ( 2 , 3 ) is a widely used technique for quantitatively and qualitatively evaluating specific protein expression. This chapter describes a Western immunoblot technique routinely used in our laboratories for protein analysis with representative data shown in Fig. 1 . The protocols for SDS-PAGE and electrophoretic transfer are based on using the Mini-PROTEAN II system (Bio-Rad, Hercules, CA). Protein detection is based on using the enhanced chemiluminescence (ECL) detection method (Amersham, Arlington Heights, IL). In this technique, mixtures of solubilized proteins under denaturing conditions (in the presence of detergent [SDS], and reducing agent [mercaptoenthanol]) are separated by electrophoresis based on their molecular weights, followed by transferring proteins to PVDF membrane and identifying a protein of interest using a specific antibody. It is highly recommended that, before beginning the protocol, the operator read Subheading 4 .   Fig. 1.  Western immunoblot analysis of protein expression of eNOS in ovine fetoplacental artery endothelial cells in culture: Proteins (2 μg protein/lane) were separated by electrophoresis (7.5% SDS-PAGE gels). After transfer to the membrane, immunoblotting was performed using a mouse monoclonal ecNOS antibody (1∶750 [0.33 μg/mL]; Transduction Laboratories, Lexington, KY) with ECL dectection at 5-min exposure, as described. Lanes 1–2, controls; lanes 3–8, cells treated with basic fibroblast growth factor (1 or 10 ng/mL) for 24 h.