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Gαi Activation Assay Kit

价格6800
品牌NewEastBio   
产地美国
货号80301
免疫原Mouse
规格20Test
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  • 概述

    Configuration-specific Monoclonal Antibody Based
    Gαi Activation Assay Kit
    Catalog Number80301

    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.


        Gαi family is the largest family of G proteins. They relay signals from many GPCRs to regualte
    various biological functions. There were no direct methods to measure the activation of Gαi proteins
    by receptors (until this assay kit). Most reports used one of the downstream pathway, i.e. the
    inhibition of adenylyl cyclases, as a readout. Alternatively, sensitivity to pertussis toxin (PTX) was

    used as an indicator of possible Gαi proteins invovled in a signaling pathway.


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

    彩票app下载官网-正规彩票投注app-彩票app官方quantities to perform 20 assays.


        NewEast Biosciences Gαi Activation Assay Kit is based on the monoclonal antibody specifically
    recognizing the active GTP-bound Gαi proteins. This monoclonal antibody has much lower affinity
    towards the inactive Gαi proteins. Therefore, after activation by receptor signals, active GTP-bound
    i proteins could be immunoprecipitated by this monoclonal antibody and further quantified by
    western blot with another anti- Gαi antibody. 


    Assay Principle


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

    precipitated active Gαi proteins will be detected by immunoblots with another anti-Gαi antibody. 



    Kit Components


    1. Anti-active Gαi, Mouse Monoclonal Antibody (Catalog No. 26901): One vial – 22 µL (1mg/mL) in PBS,     pH 7.4, contained 50% glycerol. This antibody specifically recognizes GTP- Gαi 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
        mMNaCl, 5 mM EDTA, 5% Triton X-100.

    4. Anti-Gαi, Mouse Monoclonal Antibody (Catalog No. 26003): One vial – 22 µL(1 mg/mL) in PBS, 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.

    彩票app下载官网-正规彩票投注app-彩票app官方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 per10 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.

    彩票app下载官网-正规彩票投注app-彩票app官方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. 
    Clear          the  lysates by centrifugation for 10 minutes (12,000 x g at 4 °C).

    8. 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.

    彩票app下载官网-正规彩票投注app-彩票app官方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).

    彩票app下载官网-正规彩票投注app-彩票app官方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. 


    彩票app下载官网-正规彩票投注app-彩票app官方8. Clear the lysates by centrifugation for 10 minutes (12,000 x g at 4 °C).

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


     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 Gαi
    proteins, whereas in vitro GTPγS loading could activate ~50 % of the Gαi 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 Gαi Pull-Down Assay

    彩票app下载官网-正规彩票投注app-彩票app官方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 Gαi monoclonal antibody (Cat. No. 26901) 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.

    彩票app下载官网-正规彩票投注app-彩票app官方7. Pellet the beads by centrifugation for 10 seconds at 12,000 x g.

    彩票app下载官网-正规彩票投注app-彩票app官方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.

    彩票app下载官网-正规彩票投注app-彩票app官方11. Resuspend the bead pellet in 20 µL of 2X reducing SDS-PAGE sample buffer.

    12. Boil each sample for 5 minutes.

    彩票app下载官网-正规彩票投注app-彩票app官方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-Gαi monoclonal antibody (Cat. No. 26003), freshly diluted
    1:100 ~ 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 with
    constant 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20191115102342.png


        Gαi activation assay. A. CHO cells were transfected with wild-type Gαi1 (lanes 1 and 2) or constitutively active Gαi1-Q204L (lane 3). Cell lysates were treated with GDP (lane 1) or GTPγS (lane 3). Lysates were then incubated with an anti-active Gαi monoclonal antibody (Cat. No. 26901) (top panel). The precipitated active Gαi was immunoblotted with an anti- Gαi monoclonal antibody (Cat. No. 26003). The bottom panel shows the Western blot with anti- Gαi monoclonal antibody (Cat.No.26003) of the cell lysates. B. HEK293 cells stably expressing human m2 mAChR were treated with(lane 2) or without (lane 1) carbachol. Cell lysates were then incubated with an anti-active Gαmonoclonal antibody (Cat. No. 26901) (top panel). The precipitated active Gαi was immunoblotted with an anti- Gαi rabbit polyclonal antibody (Cat. No. 21006). The bottom panel shows the Western blot with anti-tubulin of the cell lysates. 


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