Measurement of GTPγS binding to specific G proteins in membranes using G‐protein antibodies

A novel method to quantitatively measure GTPγS binding to specific G proteins in crude membranes using G‐protein antibodies was developed, which quantitatively and specifically detected the binding of G TPγS to purified G12 and G3.

Abstract

We developed a novel method to quantitatively measure GTP gamma S binding to specific G proteins in crude membranes using G-protein antibodies. The basic strategy was that the materials were initially incubated with [35S]GTP gamma S at 37 degrees C. After 4 degrees C incubation in the wells of an ELISA plate precoated with G-protein antibodies, the radioactivity of each well was counted. This method, using an anti-Gi antiserum and an anti-Gs antiserum, quantitatively and specifically detected the binding of GTP gamma S to purified Gi2 and Gs. In S49 cell membranes, GTP gamma S binding to immunoreactive Gs was observed in a time-dependent manner that obeyed first-order kinetics, and the rate constant was stimulated approximately twofold in response to isoproterenol. The effect of isoproterenol was not observed in unc mutant membranes. The present method thus makes it possible to quantitatively measure GTP gamma S binding to specific G proteins in cell membranes.