Assay Report Card

ID: ALA3888477

Type: Binding

Description: Enzyme Kinetic Assay: PTP activity was assayed using p-nitrophenyl phosphate (pNPP) as a substrate in 3,3-dimethylglutarate buffer (50 mM 3,3-dimethylglutarate, pH 7.0, 1 mM EDTA, 150 mM NaCl, 2 mM DTT, 0.1 mg/mL BSA) at 25° C. The assays were performed in 96-well plates. Normally, to determine the IC50 values, the reaction was initiated by the addition of enzyme (final concentration at 10 nM) to a reaction mixture (0.2 mL) containing 2 mM (Km for the substrate) pNPP with various concentrations of inhibitors. The reaction rate was measured using a SpectraMax Plus 384 Microplate Spectrophotometer (Molecular Devices). To determine the mode of inhibition, the reactions were initiated by the addition of PTP-MEG2 to the reaction mixtures (0.2 mL) containing various concentrations of pNPP with different concentrations of the inhibitor 7.