Applied Biochemistry and Biotechnology, Vol.162, No.4, 1065-1079, 2010
Influence of Ionic Strength, pH, and SDS Concentration on Subunit Analysis of Phycoerythrins by SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is often used for subunit analysis of proteins, but it is not efficient to make the alpha- and beta-subunits of phycoerythrins separated by normal SDS-PAGE. In this research, subunit components and subunit molecular weights of four purified phycoerythrins were analyzed by SDS-PAGE. Four factors including Tris concentration, pH, ammonium persulfate (APS), and SDS concentration were studied for their effects on SDS-PAGE of phycoerythrins. It showed that these factors can influence the separation of alpha- and beta-subunits, electrophoresis effect of gamma-subunits, apparent molecular weights of subunits, and mobility of marker proteins. The alpha- and beta-subunits separated better in the case of lower SDS concentration, lower Tris concentration, higher pH, and/or lower APS addition in separating gels. The molecular weights of alpha- and beta-subunits increased when Tris concentration increased in a certain range. It can be concluded that factors critical to subunit analysis by SDS-PAGE are SDS concentration and ionic strength, both of which are related to critical micelle concentration of SDS and ratio of SDS monomer to micelle in SDS-PAGE system. The ratio is postulated to influence SDS-PAGE by influencing the amount of SDS bound to polypeptides and shapes of polypeptide-SDS complexes.