THE DEVELOPMENT OF HIGH RESOLUTION ELECTROPHORETIC TECHNIQUES FOR THE ANALYSIS OF MEMBRANE PROTEINS UNDER DENATURING AND NON-DENATURING CONDITIONS

Techniques have been developed for performing one- and two-dimensional polyacrylamide gel electrophoresis of pigeon erythrocyte membranes under both denaturing and non-denaturing conditions. For non-denaturing procedures, pigeon erythrocyte plasma membranes were solubilized at 4.6(DEGREES)C by sonication in the presence of the zwitterionic detergent, Zwittergent 3-14 (Calbiochem-Behring, La Jolla, CA.) or the nonionic detergent, Triton X-100. Solubilization was greater than 80% with either detergent. The soluble extracts were adjusted to 0.020 M in Triton X-100, 0.099 M in Zwittergent and 0.012 M in dibutylformamide and resonicated. Isoelectric focusing was done in gel rods at 8(DEGREES)C. The survival of enzymatic activities was assessed using histochemical staining techniques. ATPase, GTPase and protease activities were tested for and all three were observed in gels of pigeon erythrocyte membrane extracts. NADH dehydrogenase was absent in focused extracts of erythrocyte membranes but detectable in focused extracts of mitochondrial membranes. Thus focusing procedure was also combined with a second dimension electrophoresis modified from the method of Davis (Annals N.Y. Acad. of Sci. (1964)), to give a two-dimension non-denaturing procedure. This technique was used to identify detergent-binding proteins in the membrane extract. The native focused gels were also electrophoresed in a second dimension with SDS (i.e., under denaturing conditions) by a procedure modified from O\u27Farrell (J. Biol. Chem. (1975) 250, 4007-4021). This modified procedure employs a combined pH and acrylamide gradient (a double gradient) in the second dimension with the addition of urea and n-butyl urea as denaturing agents. Two dimensional electrophoresis was also done under denaturing conditions in both dimensions combining a denatured isoelectric focusing gel and the double gradient second dimension