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      Rab35 Activation Assay Kit

      价格6800
      品牌NewEastBio   
      产地美国
      货号82801
      免疫原Mouse
      规格20Test
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      • 概述

        Configuration-specific Monoclonal Antibody Based

        Rab35 Activation Assay Kit

        Catalog Number:82801

        20 assays


        Product Description


        The Rab family of GTPases regulates eukaryotic vesicular membrane traffic. Rab35 is with both plasma

        membrane and endosomal localization, and has been implicated in diverse processes that include T-cell receptor recycling, oocyte yolk protein recycling and cytokinesis. Rab35 regulates neurite outgrowth in neuronal-like cells, and can induce protrusions.


        Currently there is no direct assay to measure the activation of Rab35 GTPases.


        NewEast Biosciences Rab35 Activation Assay Kit is based on the configuration-specific monoclonal

        antibody that specifically recognizes Rab35-GTP, but not Rab Rab35-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- Rab35-GTP monoclonal antibodies can also be used to monitor the activation of Rab35 in

        cells and in tissues by immunohistochemistry.


        NewEast Biosciences Rab35 Activation Assay Kit provides a simple and fast method to monitor the

        activation of Rab35. Each kit provides sufficient quantities to perform 20 assays. 


        Assay Principle


        NewEast Biosciences Rab35 Activation Assay Kit bases on the configuration-specific anti-Rab35-GTP

        monoclonal antibody to measure the active Rab35-GTP levels, either from cell extracts or from in vitro

        GTPγS loading Rab35 activation assays. Briefly, anti-active Rab35 mouse monoclonal antibody will be

        incubated with cell lysates containing Rab35-GTP. The bound active Rab35 will then be pulled down

        by protein A/G agarose. The precipitated active Rab35 will be detected by immunoblot analysis using

        anti- Rab35 rabbit polyclonal antibody. 


        Kit Components


        1. Anti-active Rab35, Mouse Monoclonal Antibody (Catalog No. 26922): One vial – 22 μL (1

            mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide. This antibody

            specifically recognizes Rab35 -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, 750

            mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.

        4. Anti- Rab35, Rabbit Polyclonal Antibody (Catalog No. 21078): 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 Rab35, whereas

            in vitro GTPγS protein loading will activate nearly 90% of the Rab35.

        1. Aliquot 0.5 ml of each cell extract to two microfuge tubes (or use 1 μg of purified Rab35 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 Rab35 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 Rab35 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- Rab35 polyclonal antibody, freshly diluted 1:50~1000

            (depending on the amount of Rab35 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 Rab35 Activation

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

        QQ截图20191118145815.png

        Rab35 activation assay. Purified GST-tagged Rab35 proteins (Cat. #10132) were immunoprecipitated

        with the anti-active Rab35 monoclonal antibody (Cat. #26922) after treated with GDP (lane 1) or GTPγS

        (lane 2), and was blotted with anti-Rab35 polyclonal antibody(Cat. #21078). Input control is shown in

        the bottom panel. 


        Related Products
        Catalog#NameSizePrice
        82801Rab35 Activation Assay Kit 20 assays ¥6800  
        26922Active Rab35-GTP Monoclonal Antibody 30 μL ¥4800   
        21078Anti-Rab35 Rabbit Polyclonal Antibody 100 μL ¥2600  



        Publications:
        1.  VASP promotes TGF-β activation of hepatic stellate cells by regulating Rab11 dependent plasma membrane targeting of TGF-β receptors
            Hepatology Volume 61, Issue 1, pages 361–374, January 2015
        2.  Rab5 Activity Regulates GLUT4 Sorting into Insulin-Responsive and Non-Insulin-Responsive Endosomal Compartments: A Potential Mechanism for Development of Insulin Resistance
            Endocrinology. 2014 Sep;155(9):3315-28



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