Gαs 活性检测试剂盒/推荐商家现货供应

Gαs Pull-Down Activation Assay Kit

Cat. # 80801

Introduction

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 regulate various biological functions such as the stimulation of adenylyl cyclases. There were no direct methods to*the activation of Gαs proteins by receptors (until this assay kit). Most reports used one downstream pathway, the increase of cAMP, as a readout.

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.

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

1. Anti-Gαs-GTP Mouse Monoclonal Antibody (Cat. # 26906): One vial – 35 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Gαs-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-Gαs Mouse monoclonal Antibody (Cat. # 26006): 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.

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

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

Gαs Activation Assay.Purified Gαs 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-Gαs-GTP monoclonal antibody (Cat. # 26906). Immunoblot was with an anti-Gαs monoclonal antibody (Cat. # 26006).

Assay Procedure

1X Assay/Lysis Buffer: Mix the 5X Stock (Cat. # 30301) briefly and dilute to 1X in deionized water. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 µg/mL leupeptin, or 10 µg/mL aprotinin.

1. Culture cells (one 10-cm plate, ~107 cells) to approximately 80-90% confluence. Stimulate the cells with activator or inhibitor as desired.

2. Aspirate the culture media and wash twice with ice-cold PBS.

3. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer (See Reagent Preparation) to the cells (0.5-1 mL per 10 cm tissue culture plate).

4. Place the culture plates on ice for 10-20 minutes.

5. Detach the cells from the plates by scraping with a cell scraper.

6. Transfer the lysates to appropriate size tubes and place on ice.

7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the genomic DNA.

8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.

9. Collect the supernatant and store the sample (~1-2 mg of total protein) on ice for immediate use, or snap freeze and store at -70°C for future use.

1. Culture cells and stimulate with activator or inhibitor as desired.

2. Perform a cell count and then pellet the cells through centrifugation.

3. Aspirate the culture media and wash twice with ice-cold PBS.

4. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer (See Reagent Preparation) to the cell pellet (0.5-1 mL per 107 cells).

5. Lyse the cells by repeated pipetting.

6. Transfer the lysates to appropriate size tubes and place them on ice.

7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the genomic DNA.

8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.

9. Collect the supernatant and store sample on ice for immediate use, or snap freeze and store at -70°C for future use.