GTPγS Binding Assay for Melatonin Receptors in Mouse Brain Tissue.

The [S]GTPγS assay is a method that measures the level of G protein activation by determining the binding of [S]GTPγS, a non-hydrolyzable and radioactively labeled GTP analog, to Gα subunit of heterotrimeric G protein upon activation of G protein-coupled receptors (GPCR). The power of this assay lies in the fact that it measures an early event of GPCR signaling in cells expressing recombinant receptors and cells and tissues expressing endogenous receptors. The present protocol describes a sensitive method for studying G protein activation by melatonin receptors MT and MT, in membranes prepared from mouse brain. Immunoprecipitation of [S]GTPγS-labeled G proteins with Gα subunit specific antibodies (Gi, Gq, etc.) allows to determine the activation of specific G proteins. The assay can be easily applied to other tissues.