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| | p-NPP Assay |
| Description: | For the 96-well plate validation assay, sodium orthovanadate (Sigma Aldrich) was utilized as a positive control for inhibition [Swarup et al., Biochem. Biophys. Res. Commun., 107:1104-9] at a final concentration of 10 μM, to completely block DUSP5(WT) enzymatic activity. All plate assays were performed in standard 96-well clearbottom plates (Thermo Scientific Nunc) with a total assay volume of 200 μL, using a SpectraMax M5 Microplate Reader (Molecular Devices). The plate validation assay was performed with replicate columns of positive control wells, negative control wells and blank wells. Blank columns contained only buffer and pNPP. Negative control (uninhibited) contained buffer, pNPP, and DUSP5PD(WT); and, positive control contained the same components,but also contained 10 μM sodium orthovanadate. The plate was then shaken and allowed to equilibrate in the spectrophotometer at 25 °C for 30 min. After incubation, 4 μL of a 50 μM enzyme stock was dispensed into appropriate wells utilizing a single-channel pipette. This produced a final enzyme concentration of1 μM. Before a read was taken, the plate was shaken for five seconds. The initial rate for the DUSP5PD(WT) reaction was linear for approximately 90 min;and, the plate was kept in the spectrophotometer at 25 °C for an additional 80 min after the kinetic read. The endpoint reading was subsequently taken at 90 min after initiation of reaction. |
| Affinity data for this assay | |
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