Gα_i Pull-Down Activation Assay Kit

Cat. # TD-80301

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 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_i 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. Alternatively, sensitivity to pertussis toxin (PTX) was used as an indicator of possible Gα_i proteins involved in a signaling pathway.

Assay Principle

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