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Gαs Pull-Down Activation Assay Kit
Cat. # CR-W80801

Background
A structurally diverse repertoire of ligands, from photons to large peptides, activates G protein-coupled receptors (GPCRs) to elicit their physiological functions. Ligand-bound GPCRs, in turn, function as guanine nucleotide exchange factors catalyzing the exchange of GDP bound on the Gα subunit with GTP in the presence of Gβγ, causing the dissociation of the Gα subunit from the Gβγ dimer to form two functional units (Gα and Gβγ). Both Gα and Gβγ subunits signal to various cellular signaling pathways. Based on the sequence and functional homologies, G proteins are grouped into four families: Gs, Gi, Gq, and G12.

Gαs family relays signals from many GPCRs to regulate various biological functions such as the stimulation of adenylyl cyclases. There were no direct methods to measure the activation of Gαs proteins by receptors (until this assay kit). Most reports used one downstream pathway, the increase of cAMP, as a readout.

Gαs Activation Assay Kit is based on the monoclonal antibody specifically recognizing the active GTP-bound Gαs proteins. This monoclonal antibody has much lower affinity towards the inactive Gαs proteins. Therefore, after activation by receptor signals, active GTP-bound Gαs proteins could be immunoprecipitated by this monoclonal antibody and further quantified by western blot with another anti-Gαs antibody.

Assay Principle
The Gαs Activation Assay Kit uses configuration-specific anti-Gαs-GTP Mouse monoclonal antibody to measure Gαs-GTP levels in cell extracts or in vitro GTPγS loading Gαs activation assays. Anti-Gαs-GTP mouse monoclonal antibody is first incubated with cell lysates containing Gαs-GTP. Next, the GTP-bound Gαs is pulled down by protein A/G agarose. Finally, the precipitated Gαs-GTP is detected through immunoblot analysis using anti-Gαs mouse monoclonal antibody.