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Gαo Pull-Down Activation Assay Kit
Cat. # CR-W80901

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 Gαo) 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 Gαo 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.

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