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).
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PTEN Phosphatase Activity Assay
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Corresponding Organization : Harvard University
Other organizations : Johns Hopkins University
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Variable analysis
independent variables
- PTEN enzyme concentration (0.5 - 1.5 μg)
- PTEN activity toward diC6-PIP3 (measured by the evolution of inorganic phosphate)
- Absorbance at 620 nm
- Reaction buffer (50 mM Tris, 10 mM BME, pH 8.0)
- Reaction temperature (30°C)
- Reaction time (10 minutes)
- DiC6-PIP3 concentration (160 μM)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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