Affinity purification of plasma membranes.
The interaction of biotin and avidin was used to affinity purify plasma membranes for use in in vitro studies of the epidermal growth factor (EGF) receptor and other cell-surface molecules. Biotinylated mouse fibroblasts were homogenized and plasma membranes purified using immobilized monomeric avidin. Capturing the membranes on the solid-phase support facilitated buffer exchange, protein analysis, and assay of receptor function. Electron microscopy and enzyme analysis showed that the plasma membranes obtained were of significantly improved purity when compared with crude membrane preparations. In particular, contamination with other cellular membranes, such as endoplasmic reticulum, mitochondria, lysosomes, and Golgi, is reduced considerably in the purified biotinylated membrane preparations. By titrating the level of biotinylation of whole cells, we identified a level of biotinylation that produces a high yield of pure cell-surface membranes but does not interfere with ligand activation of the EGF receptor protein (as determined by in vitro autophosphorylation assays).This method produces highly purified fibroblast plasma membranes quickly and with reasonable yield using standard laboratory equipment and should be easily adapted to suit experiments involving the activation of other cell surface molecules, signal transduction pathways initiated from the cell surface, and proteome analysis of plasma membranes from a wide variety of cells.
deBlaquiere
J
J
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Melbourne, Australia. burgess@ludwig.edu.au
Burgess
A W
AW