Product Description

Gs, Gi, Gq, and G12.

of cAMP, as a readout.

quantities to perform 20 assays.

Assay Principle

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.

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-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 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.

Storage

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

Materials Needed but Not Supplied

Reagent Preparation

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

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

7. If nuclear lysis occurs, the cell lysates may become very 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.

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

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

Assay Procedure

1. Aliquot 0.5 – 1 mL of cell lysate to a microcentrifuge tube.

2. Adjust the volume of each sample to 1 mL with 1X Assay/Lysis Buffer.

3. Add 1 µL anti-active Gαs monoclonal antibody (Cat. No. 26906) to the tube.

4. Thoroughly resuspend the protein A/G agarose bead slurry by vortexing or titurating.

5. Add 20 µL of resuspended bead slurry to each tube.

6. Incubate the tubes at 4 °C for 1 hour with gentle agitation.

7. Pellet the beads by centrifugation for 10 seconds at 12,000 x g.

8. Aspirate and discard the supernatant, making sure not to disturb/remove the bead pellet.

9. Wash the bead 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating each time.

10. After the last wash, pellet the beads and carefully remove all the supernatant.

11. Resuspend the bead pellet in 20 µL of 2X reducing SDS-PAGE sample buffer.

12. Boil each sample for 5 minutes.

13. Centrifuge each sample for 10 seconds at 12,000 x g. 

1. Load 20 µL/well of pull-down supernatant to a polyacrylamide gel. Also, it’s recommended
        to include a pre-stained MW standard (as an indicator of a successful transfer in step 3).

2. Perform SDS-PAGE as per the manufacturer’s instructions.

3. Transfer the gel proteins to a PVDF or nitrocellulose membrane as per the manufacturer’s

    instructions. 

1. Following the electroblotting step, immerse the PVDF membrane in 100% Methanol for 15 seconds, and
    then allow it to dry at room temperature for 5 minutes.

2. Block the membrane with 5 % non-fat dry milk or 3 % BSA in TBST for 1 hr at room temperature with
     constant agitation.

    freshly diluted in 5 % non-fat dry milk or 3 % BSA/TBST, for 1 hr at room temperature withconstant
     agitation.

6. Use the detection method of your choice. 

Example of Results

(Cat. No. 26006).