A GENERAL-METHOD FOR PLASMA-MEMBRANE ISOLATION BY COLLOIDAL GOLD DENSITY SHIFT

A general method for isolating plasma membranes is described. Vegetati ve amoebae of Dictyostelium discoideum were allowed to directly adsorb raw colloidal gold of particle diameter 10-20 nm. After quenching the gold surface, the cells were lysed and the lysates were diluted in 60 % sucrose and centrifuged through a 65% sucrose cushion to selectively pellet the gold-laden membranes. Three generally applicable exogenous cell surface markers were used to follow the plasma membranes: interc alated [H-3]cholesterol, octadecylrhodamine, and the adsorbed gold col loid itself. The isolates routinely contained similar to 60% of these tags, enriched similar to 15-fold with respect to protein. The recover y and degree of enrichment of contaminating markers in the plasma memb rane fraction were lysosomes (3% and 1-fold); mitochondria (11% and 3- fold); rough endoplasmic reticulum, as reflected by RNA (3% and 0.7-fo ld); and DNA (9% and 4-fold). Membrane proteins and lipids were quanti tatively solubilized from the gold by detergents. We conclude that thi s methodology provides an approach to the isolation of plasma membrane s which compares favorably to existing techniques with respect to yiel d, purity, and ease. (C) 1994 Academic Press, Inc.