PTEN phosphatase activity assay

PTEN activity toward a water-soluble substrate (diC6-PIP₃, from Avanti Polar Lipids) was determined by the evolution of inorganic phosphate as measured with a malachite green detection kit (R and D Biosystems). Assays were conducted as described previously¹⁶. Briefly, 0.5 – 1.5 μg PTEN was incubated with 160 μM diC6 PIP₃ for 10 minutes in 25 μl reaction buffer (50 mM Tris, 10 mM BME, pH 8.0) at 30°C. Samples were quenched with malachite green reagent (R and D Biosystems) and absorbance was measured at 620 nm.