Gαz 活性检测试剂盒/2023文献更新

Gαz Pull-Down Activation Assay Kit

Cat. # 81001

IntroductionA. Background

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 regulate various biological functions. There were no direct methods to * the activation of Gαz proteins by receptors (until this assay kit). Most reports used one of the downstream pathways, i.e. the inhibition of adenylyl cyclases, as a readout.

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 by western blot with another anti-Gαz antibody.

B. Assay Principle

The Gαz Activation Assay Kit uses configuration-specific anti-Gαz-GTP Mouse monoclonal antibody to* Gαz-GTP levels in cell extracts or in vitro GTPγS loading Gαz activation assays. Anti-Gαz-GTP mouse monoclonal antibody is first incubated with cell lysates containing Gαz-GTP. Next, the GTP-bound Gαz is pulled down by protein A/G agarose. Finally, the precipitated Gαz-GTP is detected through immunoblot analysis using anti-Gαz mouse monoclonal antibody.

C. Kit Components

1. Anti-Gαz-GTP Mouse Monoclonal Antibody (Cat. # 26908): One vial – 35 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Gαz-GTP from all vertebrates.

2. Protein A/G Agarose (Cat. # 30301): One vial – 600 µL of 50% slurry.

3. 5X Assay/Lysis Buffer (Cat. # 30302): One bottle – 30 mL of 250 mM Tris-HCl, pH 8, 750mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.

4. Anti-Gαz Mouse monoclonal Antibody (Cat. # 26011): One vial – 50 µL (1mg/mL) in PBS, pH 7.4, contained 50% glycerol.

5. 100X GTPγS (Cat. # 30303): One vial – 50 µl at 10 mM, use 5 µL of GTPγS for  GTP-labeling of 0.5 mL of cell lysate.

6. 100X GDP (Cat. # 30304): One vial – 50 µl at 100 mM, use 5 µL of GDP for GDP-labeling of 0.5 mL of cell lysate.

7. HRP-Goat Anti-Rabbit IgG (Cat. #29002): 50 µL (0.4 mg/mL) in PBS, pH 7.4, contained 50% glycerol.

D. Materials Needed but Not Supplied

1. Stimulated and non-stimulated cell lysates

2. Protease inhibitors

3. 4°C tube rocker or shaker

4. 0.5 M EDTA at pH 8.0

5. 1.0 M MgCl2

6. 2X reducing SDS-PAGE sample buffer

7. Electrophoresis and immunoblotting systems

8. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05%  Tween-20)

9. Immunoblotting blocking buffer (TBST containing 5% Non-fat Dry Milk or 3% BSA)

10. ECL Detection Reagents

E. Example Results

The following figure demonstrates example results seen with the Gαz Activation Assay Kit. For reference only.

Gαz Activation Assay.Purified Gαz proteins were loaded as a control (lanes 1) or immunoprecipitated after treated with GDP (lane 2) or GTPγS (lane 3). Immunoprecipitation was done with the anti-Gαz-GTP monoclonal antibody (Cat. # 26908). Immunoblot was with an anti- Gαz polyclonal antibody (Cat. # 26011).