Tl. Steck et M. Lavasa, A GENERAL-METHOD FOR PLASMA-MEMBRANE ISOLATION BY COLLOIDAL GOLD DENSITY SHIFT, Analytical biochemistry, 223(1), 1994, pp. 47-50
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.