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.

    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 ?L (1mg/ml) in PBS, pH 7.4, containing 50%
    glycerol and 0.05% sodium azide. This antibody specifically recognizes Rab11-GTP from all vertebrates.

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.

Storage

Store all kit components at 4?C 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 ?g/mL leupeptin, and 10 ?g/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 Rab11, whereas in vitro GTPγS protein loading will activate nearly 90% of the Rab11.

1. Aliquot 0.5 ml of each cell extract to two microfuge tubes (or use 1 ?g of purified Rab11 protein).

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

3. Add 5 ?l of 100 X GTPγS (to 100 ?M, final concentration) to one tube (positive control).

4. Add 5 ?l 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 ?l of 1 M MgCl2 (to 60 mM, final concentration).

Assay Procedure

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.

3. Add 1 ?l 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 ?L 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 ?L 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 ?L/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- 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 4^oC 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 Rab11 Activation Assay Kit. One should use the data below for reference only.

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). 

Related Products
Catalog#	Name	Size	Price
26919	Active Rab11-GTP Monoclonal Antibody	30 μL	￥4800
83201	Rab11 Activation Assay Kit	20 assays	￥6800
26030	Anti-Rab11 Mouse Monoclonal Antibody	100 μL	￥2800

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