Rab35 Pull-Down Activation Assay Kit

Cat. # 82801

Introduction

A. Background

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  the activation of Rab35 GTPases.

The Rab35 Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Rab35-GTP, but not 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.

B. Assay Principle

The Rab35 Activation Assay Kit uses configuration-specific anti-Rab35-GTP Mouse monoclonal antibody to  Rab35-GTP levels in cell extracts or in vitro GTPγS loading Rab35 activation assays. Anti-Rab35-GTP mouse monoclonal antibody is first incubated with cell lysates containing Rab35-GTP. Next, the GTP-bound Rab35 is pulled down by protein A/G agarose. Finally, the precipitated Rab35-GTP is detected through immunoblot analysis using Anti-Rab35 Rabbit Polyclonal Antibody.

C. Kit Components

1. Anti-Rab35-GTP Mouse Monoclonal Antibody (Cat. # 26922): One vial – 35 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Rab35-GTP from all vertebrates.

2. Protein A/G Agarose (Cat. # 30301): One vial – 600 µL of 50% slurry.

3. 5X Assay/Lysis Buffer (Cat. # 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 (Cat. # 21078): One vial – 50 µL (1 mg/mL) in PBS, pH 7.4, contained 50% glycerol.

5. 100X GTPγS (Cat. # 30303): One vial – 50 µl at 10 mM, use 5 µL of GTPγS for  GTP-labeling of 0.5 mL of cell lysate.

6. 100X GDP (Cat. # 30304): One vial – 50 µl at 100 mM, use 5 µL of GDP for GDP-labeling of 0.5 mL of cell lysate.

7. HRP-Goat Anti-Rabbit IgG (Cat. #29002): 50 µL (0.4 mg/mL) in PBS, pH 7.4, contained 50% glycerol.

D. 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 at pH 8.0

5. 1.0 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. ECL Detection Reagents

E. Example Results

The following figure demonstrates example results seen with the Rab35 Activation Assay Kit. For reference only.

Rab35 Activation Assay.Purified GST-tagged Rab35 proteins (Cat. # 10132) were immunoprecipitated with the anti- Rab35-GTP 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.

Assay Procedure

A. Reagent Preparation

1X Assay/Lysis Buffer: Mix the 5X Stock (Cat. # 30301) briefly and dilute to 1X in deionized water. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 µg/mL leupeptin, or 10 µg/mL aprotinin.

B. Sample Preparation

Adherent Cells

1. Culture cells (one 10-cm plate, ~107 cells) to approximately 80-90% confluence. Stimulate the 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 (See Reagent Preparation) 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 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 centrifuging at 12,000 x g and 4°C for 10 minutes.

9. Collect the supernatant and store the sample (~1-2 mg of total protein) on ice for immediate use, or snap freeze and store at -70°C for future use.

Adherent Cells

1. Culture cells and stimulate with activator or inhibitor as desired.

2. Perform a cell count and then pellet the cells through 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 (See Reagent Preparation) to the cell pellet (0.5-1 mL per 107 cells).

5. Lyse the cells by repeated pipetting.

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

7. If nuclear lysis occurs, the cell lysates may become 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 centrifuging at 12,000 x g and 4°C for 10 minutes.

9. Collect the supernatant and store sample on ice for immediate use, or snap freeze and store at -70°C for future use.

C. In vitro GTPγS/GDP Protein 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 Rab35.

1. Aliquot 0.5 mL of cell extract (or 1 µg of purified Rab35 protein) into two microcentrifuge tubes.

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

3. Positive control: add 5 µL of 100 X GTPγS (Cat. # 30302) to the 1st tube

4. Negative control: add 5 µL of 100 X GDP (Cat. # 30304) to the 2nd tube.

5. Incubate both 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 (final concentration of 60 mM).

D. Affinity Precipitation of Activated G Protein

1. Aliquot 0.5-1 mL of cell lysates (about 1 mg of total cellular protein) to a microcentrifuge tube.

2. Adjust the volume to 1 mL with 1X Assay/Lysis Buffer (See Reagent Preparation).

3. Add 1 µL anti-Rab35-GTP antibody (Cat. # 26922).

4. Prepare the protein A/G Agarose bead slurry (Cat. # 30301) by resuspending through vertexing or titrating.

5. add 20 µL of resuspended bead slurry to above tube.

6. Incubate the tube at 4°C for 1 hour with gentle agitation.

7. Pellet the beads through centrifugation at 5,000 x g for 1 min.

8. Aspirate and discard the supernatant (making sure not to disturb or remove the bead pellet.

9. Wash the beads 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating each time.

10. After the third wash, pellet the beads through centrifugation and carefully remove all the supernatant.

11. Resuspend the bead pellet in 20 µL of 2X reducing SDS- PAGE sample buffer.

12. Boil the sample for 5 minutes.

13. Centrifuge it at 5,000 x g for 10 seconds.

E. Western Blot Analysis

1. Load 15 µL/well of pull-down supernatant to a polyacrylamide gel (17%). It is recommended to include a pre-stained MW standard (as an indicator of a successful transfer in step 3 below).

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.

Note: Steps 4-11 are at room temperature with agitation

4. Following electroblotting, immerse the PVDF membrane in **** Methanol for 15 seconds, and then allow it to dry at room temperature for 5 minutes.

Note: If Nitrocellulose is used instead of PVDF, step 4 Should be skipped.

5. Block the membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hr at room temperature with constant agitation.

6. Wash the blotted membrane three times with TBST, 5 minutes each time.

7. Incubate the membrane with Anti-Rab35 Rabbit Polyclonal Antibody (Cat. # 21078), which is freshly diluted 1: 50~500 (depending on the amount of Rab35 proteins in your sample) in 5% non-fat dry milk or 3% BSA in TBST, for 1-2 hr at room temperature with constant agitation or at 4°C overnight.

8. Wash the blotted membrane three times with TBST, 5 minutes each time.

9. Incubate the membrane with a secondary antibody (Cat. # 29002), which is freshly diluted 1: 1000 in 5% non-fat dry milk or 3% BSA in TBST, for 1 hr at room temperature with constant agitation.

10. Wash the blotted membrane three times with TBST, 5 minutes each time.

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