Dic6 pip3

PTEN’s phosphatase activity was measured using the soluble PIP3 substrate, diC6PIP3 (dihexanoyl-phosphatidyl inositol 3,4,5-triphosphate, Avanti Polar Lipids), and monitoring phosphate release by the Malachite Green assay (R&D Biosystems) at 620 nm. Initial velocities were measured in a 25 μL buffered solution containing 50 mM Tris pH 8.0, 10 mM BME, 0.05 mg/mL ovalbumin, 0.5-20 μg PTEN, and varying concentrations of substrate (20-160 μM) ^{18 (link)}.

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 previously^{16 (link)}. 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.

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^{[19 (link)]} detection kit (R and D Biosystems). Assays for were conducted as described previously.^{[9 , 10 (link), 14 (link)]} 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. The data were analyzed using the Michaelis-Menten equation, v=VmS/(Km+S) using non-linear curve fitting in Prism 6 software (Graphpad).