PhastSystem electrophoresis in beta-octylglucoside containing gels with immunodetection of a nondenatured vesicle-associated membrane protein

Recombinant vesicle-associated membrane protein (rVAMP), implicated as a pa rticipant in membrane exocytosis and fusion (a "SNARE protein"), was subjec ted to gel electrophoresis in the miniaturized gels of the PhastSystem (Pha rmacia) containing the nondenaturing, nonionic detergent beta-octylglucosid e (OG), followed by immunodetection of the protein. Three major components of nondenatured rVAMP are detected by Western blotting both in 0.5% OG and in the absence of detergent. Their separation increases with increasing gel concentration above 7%T. Ferguson plot analysis indicates that the three s pecies of VAMP are size isomers (i.e., they differ in size but share a comm on surface net charge density), the common point of intersection of the plo ts (mu-point) being a measure of their common free mobility. By the criteri a of size and free mobility (related to surface net charge), VAMP component s I, II and III in 0.5% OG-containing buffer are indistinguishable from com ponents II, III and IV, respectively, observed in the absence of the deterg ent. The feasibility of immunodetection of nondenatured rVAMP on gels conta ining nondenaturing detergents opens up the possibility of gaining biochemi cal information regarding nondenatured SNARE protein complexes and SNARE pr oteins linked to membrane fragments.