Precipitation of dilute chromatographic samples (ng/ml) containing interfering substances for SDS-PAGE

Abstract

SDS-PAGE of chromatographic fractions requires prior removal of salts, detergents, denaturants, or organic solvents which may perturb the electrophoretic separation. Likewise, to successfully visualize minute amounts of protein present in chromatographic fractions, they must often be concentrated before analysis by SDS-PAGE. In this study, we used a dye precipitation procedure for simultaneous removal of interfering substances and concentration of dilute samples (ng/ml) before analysis by SDS-PAGE. Nanogram amounts of protein (143 ng) were effectively precipitated with a pyrogallol red-molybdate reagent from commonly used chromatographic buffers containing various interfering solutes or solvents. Proteins were successfully precipitated from solution in the presence of organic solvents (acetonitrile, methanol, 2-propanol), chaotropic agents (6 M urea, 6 M guanidine-HCl), a protein stabilizer (40% sucrose), metal chelators (30 mM EDTA and 30 mM EGTA), or high salt (1.0 M NaCl). Detergents, at concentrations up to twice their critical micelle concentrations, from the nonionic class (Triton X-100, Tween 20) or from the zwitterionic class (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) did not inhibit protein precipitation. Some interference was observed when proteins were precipitated in the presence of ammonium sulfate (0. 5-2.0 M). Proteins did not precipitate in the presence of ionic detergents (SDS and cetyltrimethylammonium bromide). The sensitivity of the combined pyrogallol red-molybdate precipitation/SDS-PAGE procedure is approximately 7 ng. Two other methods of precipitating proteins (trichloroacetic acid and phenol-ether) both exhibited varying degrees of effectiveness, ranging from 714 to 7 ng/ml, in the precipitation of individual proteins. In summary, the pyrogallol red-molybdate protein precipitation procedure facilitates the SDS-PAGE analysis of dilute protein samples (ng/ml) from chromatographic fractions of various compositions. The method is useful for rapid pilot-scale protein fractionation and facilitates the ongoing propensity of researchers to work with minuscule amounts of protein.