Recombinant Human PTEN Protein, CF

• 6 months from date of receipt, -70 °C as supplied.
• 3 months, -70 °C under sterile conditions after opening.

• Assay Buffer: 100 mM Tris, 2 mM DTT, 0.05% (v/v) NP-40 Substitute (Sigma, Catalog # 74385), pH 8.5
• Vesicle Buffer: 10 mM HEPES, 1 mM EGTA and 1 mg/mL BSA, pH 7.5
• Recombinant Human PTEN (rhPTEN) (Catalog # 847-PN)
• Substrate: diC₁₆PIP₃ (Echelon, Catalog # P-3916)
• Lipids: dioleoyl phosphatidylcholine (DOPC), and dioleoyl phosphatidylserine (DOPS) (Avanti Polar Lipids, Catalog # 790595)
• Malachite Green Phosphate Detection Kit (R&D Systems, Catalog # DY996)
• 96-well Clear Plate (Costar, Catalog # 92592)
• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Prepare a solution of 12.5 mg/mL DOPC, 12.5 mg/mL DOPS in Vesicle Buffer.
2. Form Phospholipid Vesicles by combining substrate and lipids in Vesicle Buffer to final concentrations of 0.3 mM diC₁₆PIP₃, 625 μg/mL DOPC, 625 μg/mL DOPS. Mix vigorously.
3. Dilute rhPTEN to 0.25 ng/μL in Assay Buffer.
4. Dilute Phospholipid Vesicles to 0.09 mM diC₁₆PIP₃, 187.5 μg/mL DOPC, 187.5 μg/mL DOPS in Assay Buffer.
5. Prepare a standard curve from the 1 M Phosphate Standard supplied in the malachite green phosphate detection kit. First, add 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for a 10 mM stock. Then, add 10 µL of the 10 mM phosphate stock to 990 µL of Assay Buffer for a 100 µM stock. (This is the first dilution to use as a standard.) Finally, perform six additional two-fold serial dilutions of the 100 µM phosphate stock.
6. Load 100 μL of each point of the curve to wells in triplicate.
7. To the experimental wells, add 80 µL of 0.25 ng/μL rhPTEN. To the control wells, add 80 µL of Assay Buffer.
8. Start the reaction by adding 20 µL of dilute Phospholipid Vesicles.
9. Incubate at 37 ºC for 15 minutes.
10. Add 20 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
11. Add 20 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
12. Read plate at 620 nm (absorbance) in endpoint mode.
13. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control wells.

• rhPTEN: 20 ng = 0.00002 mg
• Substrate: 18 µM
• Standard Curve: 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10.0 nmol