Serine threonine phosphatase assay system

Phosphatase activity of mouse brain tissues was quantitated using the Serine/Threonine Phosphatase Assay System (Promega, V2460) by measuring the dephosphorylation of a phospho-peptide, RRA(pT)VA. Briefly, mouse brain tissues were homogenized in the phosphatase storage buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, I mM EGTA, 0.5% NP-40) containing protease inhibitors. Proteins were extracted on ice with periodic vortexing for 30 min, and lysates were cleared by centrifugation at 10,000 × g for 10 min at 4°C. Two hundred µl of supernatants was passed through Sephadex G-25 column to remove the residual phosphate in the supernatants. Two µl of samples was incubated with 100 µM of phosphopeptide (RRA(pT)VA) at 37 °C for 30 min in buffer consisting of 250 mM imidazole [pH 7.2], 1 mM EGTA, 50 mM MgCl₂, 5 mM NiCl₂, 250 µg/ml calmodulin and 0.1% β-mercaptoethanol without or with 20 nM okadaic acid. To terminate the reaction, 50 µl of Molybdate dye with additive mixture was added to the mixture. The mixture was incubated for 10 min at room temperature to allow color development. Absorbance was measured at A₆₀₀ using the Synergy HT plate reader (BioTek). Phosphate released was determined by comparing absorbance to a standard phosphate curve. PP2A activity was defined as the activity inhibited by the addition of 20 nM okadaic acid to the phosphatase reaction mixture.