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 (including Gαz) 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 Gαz 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.
Bioyears Biosciences Gαz Activation Assay Kit provides a direct measurement of the activation of Gαz proteins. This is a simple and fast tool to monitor the activation of Gαz. Each kit provides sufficient quantities to perform 20 assays. Bioyears Biosciences Gαz Activation Assay Kit is based on the monoclonal antibody specifically recognizing the active GTP-bound Gαz proteins. This monoclonal antibody has much lower affinity towards the inactive Gαz proteins. Therefore, after activation by receptor signals, active GTP-bound
Gαz proteins could be immunoprecipitated by this monoclonal antibody and further quantified by western blot with another anti Gαz antibody.
Bioyears Biosciences Gαz Activation Assay Kit is an immunoprecipitation/western blot assay to measure the levels of active GTP-bound Gαz proteins, either from cell extracts or from in vitro GTPγS loaded Gαz proteins. Briefly, the anti active Gαz monoclonal antibody will specifically bind to active Gαz protein. This antibody/Gαz complex will then be pulled down by protein A/G agarose. The precipitated active Gαz proteins will be detected by immunoblots with another anti Gαz antibody.
1. Anti active Gαz, Mouse Monoclonal Antibody (Catalog No. 26908): One vial – 22 µL (1mg/mL) in PBS, pH 7.4, contained 50% glycerol. This antibody specifically recognizes GTP- Gαz 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 mM NaCl, 5 mM EDTA, 5% Triton X-100.
4. Anti Gαz, Mouse Monoclonal Antibody (Catalog No. 26011): One vial – 22 µL(1 mg/mL) in PBS, 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 of 0.5 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 0.5 mL of cell lysate.
Store all kit components at 4ºC until their expiration dates. For long-term usage (more than 6 months),please keep the antibodies at -20oC.