ARF6 (ADP-ribosylation factor 6) is a member of the ARF super-family. ARF genes encode small GTPases that increase the ADP-ribosyltransferase activity of cholera toxin and are critical for vesicular trafficking as activators of phospholipase D. Arf6 regulates membrane trafficking and functions as a regulatory molecule of phagocytosis.

Currently there is no direct assay to measure the activation of Arf6 GTPase.

The Arf6 Activation Assay Kit uses configuration-specific anti-Arf6-GTP Mouse monoclonal antibody to measure Arf6-GTP levels in cell extracts or in vitro GTPγS loading Arf6 activation assays. Anti-Arf6-GTP mouse monoclonal antibody is first incubated with cell lysates containing Arf6-GTP. Next, the GTP-bound Arf6 is pulled down by protein A/G agarose. Finally, the precipitated Arf6-GTP is detected through immunoblot analysis using Anti-Arf6 Rabbit Polyclonal Antibody.

The anti-Arf6-GTP monoclonal antibody can also be used to monitor the activation of Arf6 in cells and in tissues by immunohistochemistry.

1. Anti-Arf6-GTP Mouse Monoclonal Antibody (Cat. # 26918): One vial – 35 μL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Arf6-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-Arf6 Rabbit Polyclonal Antibody (Cat. # 21032): 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.

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

The following figure demonstrates example results seen with the Arf6 Activation Assay Kit. For reference only.