Product Highlight

• Configuration-specific monoclonal antibody that recognizes Gα_i-GTP, but not Gα_i-GDP
• The only available Gα_i activation assay kit directly measures the level of Gα_i-GTP
• Providing reliable results with consistent reproducibility than other Gα_i activation assay kits based on certain downstream signaling pathways
• Providing shorter experimental time
• Could be used to monitor the activation of Gα_i in cells and in tissues by immunohistochemistry

Product Description

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 regualte 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 pathway, 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 invovled in a signaling pathway.

NewEast Biosciences Gα_i Activation Assay Kit provides a direct measurement of the activation of Gα_i proteins. This is a simple and fast tool to monitor the activation of Gα_i. Each kit provides sufficient quantities to perform 20 assays.

NewEast Biosciences Gα_i Activation Assay Kit is based on the monoclonal antibody specifically recognizing the active GTP-bound Gα_i proteins. This monoclonal antibody has much lower affinity towards the inactive Gα_i proteins. Therefore, after activation by receptor signals, active GTP-bound Gα_i proteins could be immunoprecipitated by this monoclonal antibody and further quantified by western blot with another anti-Gα_i antibody.

 

Assay Principle

NewEast Biosciences Gα_i Activation Assay Kit is an immunoprecipitation/western blot assay to measure the levels of active GTP-bound Gα_i proteins, either from cell extracts or from in vitro GTPγS loaded Gα_iproteins. Briefly, the anti-active Gα_i monoclonal antibody will specifically bind to active Gα_i protein. This antibody/ Gα_i complex will then be pulled down by protein A/G agarose. The precipitated active Gα_i proteins will be detected by immunoblots with another anti-Gα_i antibody.