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 is the largest family of G proteins. They relay signals from many GPCRs to regualte various biological functions. There were no direct methods to measure the activation of Gαi proteins by receptors (until this assay kit). Most reports used one of the downstream pathway, i.e. the inhibition of adenylyl cyclases, as a readout. Alternatively, sensitivity to pertussis toxin (PTX) was used as an indicator of possible Gαi proteins invovled in a signaling pathway.
NewEast Biosciences Gαi Activation Assay Kit provides a direct measurement of the activation of Gαi proteins. This is a simple and fast tool to monitor the activation of Gαi. Each kit provides sufficient quantities to perform 20 assays.
NewEast Biosciences Gαi Activation Assay Kit is based on the monoclonal antibody specifically recognizing the active GTP-bound Gαi proteins. This monoclonal antibody has much lower affinity towards the inactive Gαi proteins. Therefore, after activation by receptor signals, active GTP-bound Gαi proteins could be immunoprecipitated by this monoclonal antibody and further quantified by western blot with another anti-Gαi antibody.
NewEast Biosciences Gαi Activation Assay Kit is an immunoprecipitation/western blot assay to measure the levels of active GTP-bound Gαi proteins, either from cell extracts or from in vitro GTPγS loaded Gαi proteins. Briefly, the anti-active Gαi monoclonal antibody will specifically bind to active Gαi protein. This antibody/ Gαi complex will then be pulled down by protein A/G agarose. The precipitated active Gαi proteins will be detected by immunoblots with another anti-Gαi antibody.