A Helping Hand for Your Research

Phone: 610-945-2007
Fax:

EN CH

G Protein Activation Assay Kits

PRODUCT CENTER

Gαs Activation Assay Kit

Price$737.00
BrandNewEastBio   
Place of OriginUSA
Catalog Number80801
ImmunogenMouse
Size20 Assays
Technical InformationView technical data
  • Data Sheet
  • Reference
Purchase by sweeping
  • Summary

    Configuration-specific Monoclonal Antibody Based
    Gαs Activation Assay Kit
    Catalog Number80801
    20 assays


    Product Description


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

    of cAMP, as a readout.


        NewEast Biosciences Gαs Activation Assay Kit provides a direct measurement of the activation of Gαs
    proteins. This is a simple and fast tool to monitor the activation of Gαs. Each kit provides sufficient

    quantities to perform 20 assays.


        NewEast Biosciences 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
    s proteins could be immunoprecipitated by this monoclonal antibody and further quantified by
    western blot with another anti- Gαs antibody.  


    Assay Principle


        NewEast Biosciences Gαs Activation Assay Kit is an immunoprecipitation/western blot assay to
    measure the levels of active GTP-bound Gαs proteins, either from cell extracts or from in vitro GTPγS
    loaded Gαs proteins. Briefly, the anti-active Gαs monoclonal antibody will specifically bind to active
    s protein. This antibody/Gαs complex will then be pulled down by protein A/G agarose. The

    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.


    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-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


    1. Stimulated and non-stimulated cell lysates
    2. Protease inhibitors
    3. 4 °C tube rocker or shaker
    4. 1 M MgCl2
    5. 2X reducing SDS-PAGE sample buffer
    6. Electrophoresis and immunoblotting systems
    7. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05 %Tween-20)
    8. Immunoblotting blocking buffer (TBST containing 5 % Non-fat Dry Milk or 3 % BSA)
    9. PVDF or nitrocellulose membrane
    10. Secondary Antibody
    11. ECL Detection Reagents 



    Reagent Preparation


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


    Sample Preparation


    Adherent Cells 

    1. Culture cells (one 10-cm plate, ~ 107cells) to approximately 80-90 % confluence. Stimulate 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 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 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.

    8. Clear the lysates by centrifugation for 10 minutes (12,000 x g at 4 °C).

    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.

    Suspension Cells
    1. Culture cells and stimulate with activator or inhibitor as desired.
    2. Perform a cell count, and then pellet the cells by 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 to the cell pellet (0.5 – 1
         mL per 1 x 10
    7cells).

    5. Lyse the cells by repeated pipetting.
    6. Transfer the lysates to appropriate size tubes and place on ice.
    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. 


    In vitro GTPγS/GDP Protein Loading for positive and negative controls
    Note: In vivo stimulation of cells with receptor ligands might activate ~10 % of the available Gαproteins, whereas in vitro GTPγS loading could activate ~30 % of the Gαs proteins that can be activated.
    1, Aliquot 0.5 mL of each cell extract to two microfuge tubes.
    2, To each tube, add 5 µL of 1M MgCl2 (to 10 mM final concentration).
    3, Add 5 µL of 100X GTPγS (to 100 µM, final concentration) to one tube (positive control).
    4, Add 5 µL of 100X GDP (to 1 mM, final concentration) to the second tube (negative control).
    5, Incubate the tubes at 30°C for 90 minutes with agitation.  


    Assay Procedure


    I. Active Gαs Pull-Down Assay

    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. 


    II. Electrophoresis and Transfer

    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. 


    III. Immunoblotting and Detection (all steps are at room temperature, with agitation)

    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.

    Note: If Nitrocellulose is used instead of PVDF, this step should be skipped.

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

        Incubate the membrane with anti-Gαs monoclonal antibody (Cat. No. 26006), freshly diluted
        1:500 ~ 1000 in 5 % non-fat dry milk or 3 % BSA/TBST, for 1-2 hr at room temperature with
        constant agitation.
    Note: To conserve antibody, incubations should be performed in a plastic bag.
    3. Wash the blotted membrane three times with TBST, 5 minutes each time.
    4. Incubate the membrane with a secondary antibody (e.g. goat anti-mouse IgG, HRP-conjugate),

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

    5. Wash the blotted membrane three times with TBST, 5 minutes each time.

    6. Use the detection method of your choice. 


    Example of Results


    The following figure demonstrates typical results seen with NewEast Biosciences Gαi Activation
    Assay Kit. One should use the data below for reference only. 

    QQ截图20191118145815.png

    Gαs activation assay. Purified Gααααs 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-active
    Gαs monoclonal antibody (Cat. No. 26906). Immunoblot was with an anti- Gαs monoclonal antibody

    (Cat. No. 26006). 


    Related Products
    Catalog#NameSizePrice
    80801s Activation Assay Kit 20 assays $737
    26906Active GαGTP Monoclonal Antibody 30 μL $495
    26006Anti Gαs Mouse Monoclonal Antibody 100 μL $239
    21007Anti Gαs Rabbit Polyclonal Antibody 100 μL $219
    Publications:
    1.  Glucocorticoid acts on a putative G protein-coupled receptor to rapidly regulate the activity of NMDA receptors in hippocampal neurons
        Am J Physiol Endocrinol Metab. 2012 Apr 1;302(7):E747-58
    2.  CRH activation of different signaling pathways results in differential calcium signaling in human pregnant myometrium before and during labor
        J Clin Endocrinol Metab. 2012 Oct;97(10):E1851-61
    3.  Estrogenic action on arterial smooth muscle: permissive for maintenance of CRHR2 expression
        Endocrinology. 2012 Apr;153(4):1915-24
    4.  Corticotropin-releasing hormone stimulates mitotic kinesin-like protein 1 expression via a PLC/PKC-dependent signaling pathway in hippocampal neurons
        Mol Cell Endocrinol. 2012 Oct 15;362(1-2):157-64
    5.   Rapid actions of xenoestrogens disrupt normal estrogenic signaling
         Steroids Volume 81, March 2014, Pages 36–42
    6.   Neuronal NF1/RAS regulation of cyclic AMP requires atypical PKC activation
         Hum. Mol. Genet. (2014)