Gαo 活性检测试剂盒/采购推荐

Gαo Pull-Down Activation Assay Kit

Cat. # 80901

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 Gαo) 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 Gαo 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.

B. Assay Principle

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

C. Kit Components

1. Anti-Gαo-GTP Mouse Monoclonal Antibody (Cat. # 26907): One vial – 35 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Gαo-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-GαoMouse monoclonal Antibody (Cat. # 21015): 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 GαoActivation Assay Kit. For reference only.

Gαo Activation Assay. Purified Gαo 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-Gαo-GTP monoclonal antibody (Cat. # 26907). Immunoblot was with an anti-Gαo polyclonal antibody (Cat. # 21015).