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Rab11 Activation Assay Kit

价格6800
品牌NewEastBio   
产地美国
货号83201
免疫原Mouse
规格20Test
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  • 概述

    Configuration-specific Monoclonal Antibody Based

    Rab11 Activation Assay Kit

    Catalog Number: 83201

    20 assays



    Product Description


        Small GTPases are a super-family of cellular signaling regulators. Rab11 has been shown to control traffic through the recycling endosome. Currently there is no direct assay to measure the activation of Rab11 GTPases.


    彩票app下载官网-正规彩票投注app-彩票app官方    NewEast Biosciences Rab11 Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Rab11-GTP, but not Rab11-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- Rab11-GTP monoclonal antibodies can also be used to monitor the activation of Rab11 in cells and in tissues by immunohistochemistry. NewEast Biosciences Rab11 Activation Assay Kit provides a simple and fast method to monitor the

    activation of Rab11. Each kit provides sufficient quantities to perform 20 assays.


    Assay Principle


        NewEast Biosciences Rab11 Activation Assay Kit bases on the configuration-specific anti- Rab11-GTP monoclonal antibody to measure the active Rab11-GTP levels, either from cell extracts or from in vitro GTPγS loading Rab11 activation assays. Briefly, anti-active Rab11 mouse monoclonal antibody will be incubated with cell lysates containing Rab11-GTP. The bound active Rab11 will then be pulled down by protein A/G agarose. The precipitated active Rab11 will be detected by immunoblot analysis using an anti-Rab11 rabbit polyclonal antibody. 



    Kit Components


    1. Anti-active Rab11, Mouse Monoclonal Antibody (Catalog No. 26919): One vial – 22 µL (1mg/ml) in PBS, pH 7.4, containing 50%
        glycerol and 0.05% sodium azide. This antibody specifically recognizes Rab11-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- Rab11, Rabbit Polyclonal Antibody (Catalog No. 21157): One vial – 22 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 forGDP-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

    3. 4 °C tube rocker or shaker

    彩票app下载官网-正规彩票投注app-彩票app官方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

    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

    彩票app下载官网-正规彩票投注app-彩票app官方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)

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

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

    彩票app下载官网-正规彩票投注app-彩票app官方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 Rab11, whereas in vitro GTPγS protein loading will activate nearly 90% of the Rab11.

    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 Rab11 protein).

    彩票app下载官网-正规彩票投注app-彩票app官方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).

    彩票app下载官网-正规彩票投注app-彩票app官方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.

    彩票app下载官网-正规彩票投注app-彩票app官方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


    彩票app下载官网-正规彩票投注app-彩票app官方I. Active Rab11 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.

    彩票app下载官网-正规彩票投注app-彩票app官方3. Add 1 µl anti-active Rab11 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.

    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 1 min at 5,000 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 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.


    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.

    彩票app下载官网-正规彩票投注app-彩票app官方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- Rab11 polyclonal antibody, freshly diluted 1:50~1000

        (depending on the amount of Rab11 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 4o彩票app下载官网-正规彩票投注app-彩票app官方C overnight.

    彩票app下载官网-正规彩票投注app-彩票app官方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.

    6. Use the detection method of your choice such as ECL. 


    Example of Results

    彩票app下载官网-正规彩票投注app-彩票app官方The following figure demonstrates typical results seen with NewEast Biosciences Rab11 Activation Assay Kit. One should use the data below for reference only.

    QQ截图20191118145815.png

    彩票app下载官网-正规彩票投注app-彩票app官方Rab11 activation assay. Purified Rab11 proteins were immunoprecipitated after treated with GDP (lane 1) or GTPγS (lane 2). Immunoprecipitation was done with the anti-active Rab11 monoclonal antibody (Cat. No. 26919). Immunoblot was with an anti-Rab11 polyclonal antibody (Cat. No. 21157). 


    Publications:
    1.  
        Hepatology Volume 61, Issue 1, pages 361–374, January 2015
    2.  
        Endocrinology. 2014 Sep;155(9):3315-28


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