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

AbstractWe developed a novel method to quantitatively measure GTPγS binding to specific G proteins in crude membranes using G-protein antibodies. The basic strategy was that the materials were initially incubated with [35S]GTPγS at 37°C. After 4°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-G3 antiserum, quantitatively and specifically detected the binding of GTPγS to purified G12 and G3. In S49 cell membranes, GTPγS binding to immunoreactive G3 was observed in a time-dependent manner that obeyed first order kinetics, and the rate constant was stimulated ∼tworold 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 GTPγS binding to specific G proteins in cell membranes