Rab11 活性检测试剂盒

Rab11 Pull-Down Activation Assay Kit

Cat. # 83201

IntroductionA. Background

Small GTPases are a super-family of cellular signaling regulators. Rab11 has been shown to control traffic through the recycling endosome.

The 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.

Currently there is no direct assay to* the activation of Rab11 GTPases.

B. Assay Principle

The Rab11 Activation Assay Kit uses configuration-specific anti-Rab11-GTP Mouse monoclonal antibody to* Rab11-GTP levels in cell extracts or in vitro GTPγS loading Rab11 activation assays. Anti-Rab11-GTP mouse monoclonal antibody is first incubated with cell lysates containing Rab11-GTP. Next, the GTP-bound Rab11 is pulled down by protein A/G agarose. Finally, the precipitated Rab11-GTP is detected through immunoblot analysis using Anti-Rab11 Rabbit Polyclonal Antibody.

C. Kit Components

1. Anti-Rab11-GTP Mouse Monoclonal Antibody (Cat. # 26919): One vial – 35 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Rab11-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-Rab11 Rabbit Polyclonal Antibody (Cat. # 21157): 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 Rab11 Activation Assay Kit. 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-Rab11-GTP monoclonal antibody (Cat. # 26919). Immunoblot was with an anti-Rab11 polyclonal antibody (Cat. # 21157).