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Gαz Pull-Down Activation Assay Kit
Cat. # CR-W81001

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.
Gαi family (including Gαz) is the largest family of G proteins. They relay signals from many GPCRs to regulate various biological functions. There were no direct methods to measure the activation of Gαz proteins by receptors (until this assay kit). Most reports used one of the downstream pathways, i.e. the inhibition of adenylyl cyclases, as a readout.
Gαz Activation Assay Kit is based on the monoclonal antibody specifically recognizing the active GTP-bound Gαz proteins. This monoclonal antibody has much lower affinity towards the inactive Gαz proteins. Therefore, after activation by receptor signals, active GTP-bound Gαz proteins could be immunoprecipitated by this monoclonal antibody and further quantified by western blot with another anti-Gαz antibody.

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