Measurement of GTPγS binding to specific G proteins in membranes using G‐protein antibodies
- 29 June 1992
- journal article
- Published by Wiley in FEBS Letters
- Vol. 305 (2) , 125-128
- https://doi.org/10.1016/0014-5793(92)80878-k
Abstract
We 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.Keywords
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