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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

    彩票app下载官网-正规彩票投注app-彩票app官方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.


    彩票app下载官网-正规彩票投注app-彩票app官方    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):彩票app下载官网-正规彩票投注app-彩票app官方 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):彩票app下载官网-正规彩票投注app-彩票app官方 One vial – 100 µL (1 mg/ml)

    彩票app下载官网-正规彩票投注app-彩票app官方    in PBS, pH 7.4, contained 50% glycerol.

    5. 100 X GTPγS (Catalog No. 30303):彩票app下载官网-正规彩票投注app-彩票app官方 One vial –100 µl at 10 mM, use 5 µL of GTPγS for

    彩票app下载官网-正规彩票投注app-彩票app官方    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

    彩票app下载官网-正规彩票投注app-彩票app官方1. Stimulated and non-stimulated cell lysates

    2. Protease inhibitors

    彩票app下载官网-正规彩票投注app-彩票app官方3. 4 °C tube rocker or shaker

    4. 0.5 M EDTA, pH8.0

    彩票app下载官网-正规彩票投注app-彩票app官方5. 1 M MgCl2

    彩票app下载官网-正规彩票投注app-彩票app官方6. 2X reducing SDS-PAGE sample buffer

    彩票app下载官网-正规彩票投注app-彩票app官方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)

    彩票app下载官网-正规彩票投注app-彩票app官方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


    彩票app下载官网-正规彩票投注app-彩票app官方• 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.

    彩票app下载官网-正规彩票投注app-彩票app官方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

    彩票app下载官网-正规彩票投注app-彩票app官方    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.

    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 107cells).

    5. Lyse the cells by repeated pipetting.

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

    彩票app下载官网-正规彩票投注app-彩票app官方7. If nuclear lysis occurs, the cell lysates may become very viscous and difficult to pipette. If this

    彩票app下载官网-正规彩票投注app-彩票app官方    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 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

    彩票app下载官网-正规彩票投注app-彩票app官方vitro GTPγS protein loading will activate nearly 90% of the RheB.

    彩票app下载官网-正规彩票投注app-彩票app官方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).

    彩票app下载官网-正规彩票投注app-彩票app官方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).

    彩票app下载官网-正规彩票投注app-彩票app官方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

    彩票app下载官网-正规彩票投注app-彩票app官方    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.

    彩票app下载官网-正规彩票投注app-彩票app官方4. Thoroughly resuspend the protein A/G Agarose bead slurry by vortexing or titurating.

    彩票app下载官网-正规彩票投注app-彩票app官方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.

    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.

    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).

    彩票app下载官网-正规彩票投注app-彩票app官方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.

    彩票app下载官网-正规彩票投注app-彩票app官方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.

    彩票app下载官网-正规彩票投注app-彩票app官方5. Wash the blotted membrane three times with TBST, 5 minutes each time.

    彩票app下载官网-正规彩票投注app-彩票app官方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

    彩票app下载官网-正规彩票投注app-彩票app官方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). 


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