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 participant in membrane exocytosis and fusion (a "SNARE protein"), was subjected to gel electrophoresis in the miniaturized gels of the PhastSystem (Pharmacia) containing the nondenaturing, nonionic detergent beta-octylglucoside (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 species of VAMP are size isomers (i.e., they differ in size but share a common surface net charge density), the common point of intersection of the plots (mu-point) being a measure of their common free mobility. By the criteria of size and free mobility (related to surface net charge), VAMP components I, II and III in 0.5% OG-containing buffer are indistinguishable from components II, III and IV, respectively, observed in the absence of the detergent. The feasibility of immunodetection of nondenatured rVAMP on gels containing nondenaturing detergents opens up the possibility of gaining biochemical information regarding nondenatured SNARE protein complexes and SNARE proteins linked to membrane fragments.
Buzás
Z
Z
Section of Macromolecular Analysis, Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1580, USA.
Kolosova
I
I
Chrambach
A
A
D000818:Animals; D003902:Detergents / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation* / Q000379:methods*; D005960:Glucosides / Q000032:analysis* / Q000737:chemistry*; D015151:Immunoblotting; D008565:Membrane Proteins / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis; D012617:Sea Urchins / Q000737:chemistry; D013696:Temperature
D003902:Detergents; D005960:Glucosides; D008565:Membrane Proteins; D011994:Recombinant Proteins; C018619:octyl-beta-D-glucoside