Gαo Pull-Down Activation Assay

Gα_o Pull-Down Activation Assay Kit

Cat. # TD-80901

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.
Gα_i family (including Gα_o) 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 Gα_o 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 Gα_oActivation Assay Kit uses configuration-specific anti-Gα_o-GTP Mouse monoclonal antibody to measure Gα_o-GTP levels in cell extracts or in vitro GTPγS loading Gα_oactivation assays. Anti-Gα_o-GTP mouse monoclonal antibody is first incubated with cell lysates containing Gα_o-GTP. Next, the GTP-bound Gα_ois pulled down by protein A/G agarose. Finally, the precipitated Gα_o-GTP is detected through immunoblot analysis using anti-Gα_omouse monoclonal antibody.