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      RheB Activation Assay Kit

      价格6800
      品牌NewEastBio   
      产地美国
      货号81201
      免疫原Mouse
      规格20Test
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      • 概述

        Configuration-specific Monoclonal Antibody Based

        RheB Activation Assay Kit

        Catalog Number:81201

        20 assays


        Product Description


            Small GTPases are a super-family of cellular signaling regulators. RheB is a member of the Ras-superfamily GTPases. RheB has been shown to interact with C-Raf, Mammalian target of rapamycin (mTOR), TSC2, Ataxia telangiectasia mutated (ATM), KIAA1303 and Ataxia telangiectasia and Rad3 related. Currently there is no direct assay to measure the activation of RheB GTPases.


            NewEast Biosciences RheB Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes RheB-GTP, but not RheB-GDP. Given the high affinity of monoclonal antibodies to their antigens, the activation assay could be performed in a short time. This assay provides the reliable results with consistent reproducibility. These anti-RheB-GTP monoclonal antibodies can also be used to monitor the activation of RheB in cells and in tissues by immunohistochemistry.


            NewEast Biosciences RheB Activation Assay Kit provides a simple and fast method to monitor the activation of RheB. Each kit provides sufficient quantities to perform 20 assays.


        Assay Principle


        NewEast Biosciences RheB Activation Assay Kit bases on the configuration-specific anti-RheB-GTP monoclonal antibody to measure the active RheB-GTP levels, either from cell extracts or from in vitro GTPγS loading RheB activation assays. Briefly, anti-active RheB mouse monoclonal antibody will be incubated with cell lysates containing RheB-GTP. The bound active RheB will then be pulled down by protein A/G agarose. The precipitated active RheB will be detected by immunoblot analysis using anti RheB rabbit polyclonal antibody. 


        Kit Components

        1. Anti-active RheB, Mouse Monoclonal Antibody (Catalog No. 26910): One
            vial – 22 μL (1mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide.     This antibody specifically recognizes RheB-GTP 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. 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-RheB, Rabbit Polyclonal Antibody (Catalog No. 21098): One vial – 100 μL (1 mg/ml)

            in PBS, pH 7.4, contained 50% glycerol.

        5. 100 X GTPγS (Catalog No. 30303): 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. 0.5 M EDTA, pH8.0

        5. 1 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. PVDF or nitrocellulose membrane

        11. Secondary Antibody

        12. 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 27?-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 107cells).

        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 27?-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 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 will activate approximately 10% of the available RheB, whereas in

        vitro GTPγS protein loading will activate nearly 90% of the RheB.

        1, Aliquot 0.5 ml of each cell extract to two microfuge tubes (or use 1 μg of purified RheB

            protein).

        2, To each tube, add 20 μl of 0.5 M EDTA (to 20 mM final concentration).

        3, Add 5 μl of 100 X GTPγS (to 100 μM, final concentration) to one tube (positive control).

        4, Add 5 μl of 100 X GDP (to 1 mM, final concentration) to the second tube (negative
            control).

        5, Incubate the tubes at 30°C for 30 minutes with agitation.

        6, Stop loading by placing the tubes on ice and adding 32.5 μl of 1 M MgCl2 (to 60 mM, final

            concentration). 


        Assay Procedure


        I. Active RheB 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 RheB monoclonal antibody to the tube.

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

        5. Quickly 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 1 min at 5,000 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 5,000 x g. 


        II. Electrophoresis and Transfer

        1. Load 15 μL/well of pull-down supernatant to a polyacrylamide gel (17%). 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 following the manufacturer’s instructions.

        3. Transfer the gel proteins to a PVDF or nitrocellulose membrane following 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-RheB rabbit
            polyclonal antibody, freshly diluted 1:50~1000 (depending on the amount of RheB
            proteins in your samples) in 5% non-fat dry milk or 3% BSA/TBST, for 1-2 hr at room
            temperature with constant agitation or at 4oC overnight.

        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-Rabbit IgG, HRP-
            conjugate),freshly diluted 1:1000 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 such as ECL. 


        Example of Results


        The following figure demonstrates typical results seen with NewEast Biosciences RheB Activation

        Assay Kit. One should use the data below for reference only. 

        QQ截图20191118145815.png

        RheB activation assay. Purified RheB proteins were immunoprecipitated after treated with GDP (lane 1) or GTPγS (lane 2). Immunoprecipitation was done with the anti-active RheB monoclonal antibody (Cat. No. 26910). Immunoblot was with an anti-RheB rabbit polyclonal antibody(Cat. No.21098). 

        Related Products
        Catalog#NameSizePrice
        26910Active RheB-GTP Monoclonal Antibody 30 μL ¥4800
        81201RheB Activation Assay Kit 20 assays ¥6800
        26015Anti-RheB Mouse Monoclonal Antibody 100 μL ¥2800
        21098Anti-RheB Rabbit Polyclonal Antibody 100 μL ¥2600
        Publications:
        1.  Inducible Nitric Oxide Synthase Drives mTOR Pathway Activation and Proliferation of Human Melanoma by Reversible Nitrosylation of TSC2
            Cancer Res. 2014 Feb 15;74(4):1067-78
        2.  SynGAP Regulates Protein Synthesis and Homeostatic Synaptic Plasticity in Developing Cortical Networks
            PLoS One. 2013 Dec 31;8(12):e83941


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