Product Highlight

• Configuration-specific monoclonal antibody that recognizes Gα_s-GTP, but not Gα_s-GDP
• The only available Gα_s activation assay kit directly measures the level of Gα_s-GTP
• Providing reliable results with consistent reproducibility than other Gα_s activation assay kits based on certain downstream signaling pathways
• Providing shorter experimental time
• Could be used to monitor the activation of Gα_s 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_s family relays signals from many GPCRs to regualte various biological functions such as the stimulation of adenylyl cyclases. There were no direct methods to measure the activation of Gα_s proteins by receptors (until this assay kit). Most reports used one downstream pathway, the increase of cAMP, as a readout.

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

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

 

Assay Principle

NewEast Biosciences Gα_s Activation Assay Kit bases on the configuration-specific anti-Gα_s-GTP monoclonal antibody to measure the active Gα_s-GTP levels, either from cell extracts or from in vitro GTPγS loading Gα_s activation assays. Briefly, anti-active Gα_smouse monoclonal antibody will be incubated with cell lysates containing Gα_s-GTP. The bound active Gα_s will then be pulled down by protein A/G agarose. The precipitated active Gα_s will be detected by immunoblot analysis using anti- Gα_s rabbit polyclonal antibody.

Publications:

1.  Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons     Am J Physiol Endocrinol Metab. 2012 Apr 1;302(7):E747-58

2.  CRH activation of different signaling pathways results in differential calcium signaling in human pregnant myometrium before and during labor     J Clin Endocrinol Metab. 2012 Oct;97(10):E1851-61

3.  Estrogenic action on arterial smooth muscle: permissive for maintenance of CRHR2 expression     Endocrinology. 2012 Apr;153(4):1915-24

4.  Corticotropin-releasing hormone stimulates mitotic kinesin-like protein 1 expression via a PLC/PKC-dependent signaling pathway in hippocampal neurons     Mol Cell Endocrinol. 2012 Oct 15;362(1-2):157-64

5.   Rapid actions of xenoestrogens disrupt normal estrogenic signaling      Steroids Volume 81, March 2014, Pages 36–42

6.   Neuronal NF1/RAS regulation of cyclic AMP requires atypical PKC activation      Hum. Mol. Genet. (2014)