Rab3 活性检测试剂盒/现货kit

Rab3 Pull-Down Activation Assay Kit

Cat. # 82601

IntroductionA. Background

Rab3 is a member of the Rab-family GTPases and is specifically targeted to synaptic vesicles Currently there is no direct assay to * the activation of Rab3 GTPases.

The Rab3 Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Rab3-GTP, but not Rab3-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 Rab3 GTPases.

B. Assay Principle

The Rap1 Activation Assay Kit uses configuration-specific anti-Rab3-GTP Mouse monoclonal antibody to* Rab3-GTP levels in cell extracts or in vitro GTPγS loading Rab3 activation assays. Anti-Rab3-GTP mouse monoclonal antibody is first incubated with cell lysates containing Rab3-GTP. Next, the GTP-bound Rab3 is pulled down by protein A/G agarose. Finally, the precipitated Rab3-GTP is detected through immunoblot analysis using Anti-Rab3A Rabbit Polyclonal Antibody.

C. Kit Components

1. Anti-Rab3-GTP Mouse Monoclonal Antibody (Cat. # 26920): One vial – 35 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Rab3-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-Rab3A Rabbit Polyclonal Antibody (Cat. # 21041): 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 Rab3 Activation Assay Kit. For reference only.

Rab3A Activation Assay.Purified GST-tagged Rab3A proteins (Cat. #10128) were immunoprecipitated with the anti-Rab3-GTP monoclonal antibody (Cat. #. 26920) after treated with GDP (lane 1) or GTPγS (lane 2), and was blotted with anti-Rab3A polyclonal antibody (Cat. # 21041). Input control is shown in the bottom panel.