Rapid isolation of plasmalemma from cultured A431 cells: characterization of epidermal growth factor receptors

• Published in: Analytical biochemistry Vol. 168; no. 2; p. 300
• Main Authors: Lin, P H, Selinfreund, R H, Wakshull, E, Wharton, W
• Format: Journal Article
• Language: English
• Published: United States 01-02-1988
• Subjects: Angiotensin II - analysis; Cell Membrane - analysis; ErbB Receptors - analysis; Humans; Phosphorylation; Tumor Cells, Cultured
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(88)90322-3

A rapid method for the purification of plasma membrane from a relatively small number of A431 cells is described. The method is a simple, two-step differential centrifugation in the presence of Ca2+ that requires a total centrifugation time of 7 min. The membrane preparations contained a high level of epidermal growth factor (EGF) receptor activity demonstrated by both the quantity of specific ligand binding and the amount of EGF-dependent phosphorylation of the receptor and an exogenous substrate. EGF-dependent autophosphorylation identified the EGF receptor in the purified membranes as an undegraded 170-kDa protein.