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 (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.
Protocol detail
PTEN Lipid Phosphatase Activity Assay
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Corresponding Organization :
Other organizations : Johns Hopkins University, Johns Hopkins Medicine, University of California, San Francisco
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Variable analysis
independent variables
- PTEN amount (0.5 - 1.5 μg)
- Inorganic phosphate production (measured by absorbance at 620 nm)
- Reaction buffer (50 mM Tris, 10 mM BME, pH 8.0)
- Substrate concentration (160 μM diC6 PIP3)
- Reaction time (10 minutes)
- Reaction temperature (30°C)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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