| Assay Method Information | |
| | In Vitro Phosphatase Assay (pNPP) |
| Description: | Briefly, assays without and with inhibitors were performed in Greiner 96-well clear bottom plates with a total assay volume of 200 μL. The assay buffer contained 100 mM Tris, 100 mM NaCl, 5 mM MgCl2 • 6H2O and 1 mM dithiothreitol (DTT) at pH 7.5. p-nitrophenyl phosphate (pNPP, Sigma Aldrich) 5 mM was used as the substrate. DUSP5 PD(WT) dephosphorylates pNPP to yield p-nitrophenolate, which absorbs at 405 nm, having an extinction coefficient of 18,000 M^-1 cm^-1. The reaction was initiated by the addition of 4 μL of a 29 μM DUPSP5 PD(WT) enzyme stock and absorbance was monitored at 25 °C over time using a Spectramax M5 microplate reader (Molecular Devices). Blanks contained only buffer and pNPP. Negative controls (without inhibitor) contained assay buffer with pNPP and DUSP5 PD(WT). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |