Rapid and effective transfer of integral membrane proteins from isoelectric focusing gels to nitrocellulose membranes

Abstract

A method describing the rapid and effective transfer of integral membrane protein from isoelectric focusing gels to nitrocellulose is described. Initial experiments were carried out with detergent-solubilized extracts of human erythrocyte membrane proteins. The effectiveness of the transfer was demonstrated by assaying for erythrocyte glucose transporter, an integral membrane protein, using specific antibodies followed by 125I-protein A and autoradiography. Several detergents including octyl glucoside, Triton X-100 and CHAPS were used in this study but only octyl glucoside effectively solubilized the glucose transporter and did not interfere with the electrotransfer of the protein. The glucose transporter separated on isoelectric focusing gels was effectively transferred after 2 h of electroblotting and was found to have an apparent pI of 6.4–6.5. These findings were substantiated by photolabeling red cell membranes with [ 3H]cytochalasin B in the presence or absence of d-glucose (which inhibits [ 3H]cytochalasin B binding to the glucose transporter) and separating the labeled proteins by two dimensional electrophoresis. With this procedure we identified a d-glucose sensitive 50–60 kDa protein focusing with an apparent pI of around pH 6.4–6.5.