Kit Components

1. Anti active Arf6, Mouse Monoclonal Antibody (Catalog No. 26918): One vial – 22 μL (1

mg/ml) in PBS, pH 7.4, containing 50% glycerol and 0.05% sodium azide. This antibody

specifically recognizes Arf6 GTP from all vertebrates.

2. Protein A/G Agarose (Catalog No. 30301): One vial – 400 μL of 50% slurry.

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.

4. Anti Arf6, Rabbit Polyclonal Antibody (Catalog No. 21032): One vial – 100 μL (1 mg/ml)

in PBS, pH 7.4, contained 50% glycerol.

5. 100 X GTPγS (Catalog No. 30303): One vial –100 μl at 10 mM, use 5 μL of GTPγS for GTP-abeling of 0.5 mL of cell lysate.

6. 100 X GDP (Catalog No. 30304): One vial –100 μl at 100 mM, use 5 μL of GDP for GDP-    labeling of 0.5 mL of cell lysate.

Storage

Store all kit components at 4oC 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 μg/mL leupeptin, and 10     μg/mL aprotinin.
Note:
For research use only .