A quantitative dot-blot immunoassay for integral membrane proteins: Preparation of pancreatic plasma membranes containing apical and basolateral domains

Abstract

Modifications of standard dot-blot immunoassay techniques have been made for the quantitation of integral membrane protein antigens. The modification involves the signal development step, which is performed by using punched-out wells of the solid support (nitrocellulose) to yield a soluble colored reaction product. This approach avoids the inhomogeneity of an insoluble reaction product and the subsequent quantitation problems encountered when such product is scanned by densitometry. The method was validated by comparing the purification and overall recoveries of a known plasma membrane enzymatic marker with integral membrane antigens of defined localization during subcellular fractionation of mouse pancreas. The final plasma membrane fraction contained both basolateral (~20-fold enriched) and apical membrane (~4-fold enriched) domains.