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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.

Gαs 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 Gαs

proteins by receptors (until this assay kit). Most reports used one downstream pathway, the increase

of cAMP, as a readout.

NewEast Biosciences Gαs Activation Assay Kit provides a direct measurement of the activation of Gαs

proteins. This is a simple and fast tool to monitor the activation of Gαs. Each kit provides sufficient

quantities to perform 20 assays.

NewEast Biosciences Gαs Activation Assay Kit is based on the monoclonal antibody specifically

recognizing the active GTP-bound Gαs proteins. This monoclonal antibody has much lower affinity

towards the inactive Gαs proteins. Therefore, after activation by receptor signals, active GTP-bound

Gαs proteins could be immunoprecipitated by this monoclonal antibody and further quantified by

western blot with another anti Gαs antibody.

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Assay Principle

NewEast Biosciences Gαs Activation Assay Kit is an immunoprecipitation/western blot assay to

measure the levels of active GTP-bound Gαs proteins, either from cell extracts or from in vitro GTPγS

loaded Gαs proteins. Briefly, the anti active Gαs monoclonal antibody will specifically bind to active

Gαs protein. This antibody/Gαs complex will then be pulled down by protein A/G agarose. The

precipitated active Gαs proteins will be detected by immunoblots with another anti Gαs antibody.

Kit Components

1. Anti active Gαs, Mouse Monoclonal Antibody (Catalog No. 26906): One vial – 22 µL (1mg/mL) in PBS,

pH 7.4, contained 50% glycerol. This antibody specifically recognizes GTP- Gαs from all vertebrates.

2. Protein A/G Agarose (Catalog No. 30301): One vial – 400 µL of 50% slurry.

3. 5X Assay/Lysis Buffer (Catalog No. 30303): One bottle – 30 mL of 250 mM Tris-HCl, pH 7.4,750 mMNaCl, 5 mM EDTA, 5% Triton X-100.

4. Anti Gαs, Mouse Monoclonal Antibody (Catalog No. 26006): One vial – 22 µL(1 mg/mL) inPBS, pH 7.4,contained 50% glycerol.

5. 100 X GTPγS (Catalog No. 30302): One vial –100 µL at 10 mM, use 5 µL of GTPγS for GTP-labeling of0.5 mL of cell lysate.

6. 100 X GDP (Catalog No. 30304): One vial –100 µL at 100 mM, use 5 µL of GDP for GDP-labeling of 0.5mL of cell lysate.

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Storage

Store all kit components at 4ºC until their expiration dates.

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Note

For Research Use Only.