| Solution Information | help | |
| Enzyme: | Dual specificity protein phosphatase 3 | |
| inhibitor: | BDBM34905 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay materials: 1) VHR1 was obtained from the assay provider's laboratory. 2) Assay Buffer: 20mM Bis-Tris pH 6.0, 1mM DTT, 0.005% Tween-20 3) Biomol Green - Biomol (Cat #AK111-1000) uHTS Procedure 1) Dispense 1.5ul of assay buffer into columns 1 and 2 of a black, clear bottom Corning (#3891) 1536 well assay plate. 2) Add 1.5ul of 1474uM pNPP in assay buffer to all wells 3) Using a HighRes biosolutions pintool dispense 20nl of 2mM compounds in DMSO to columns 5-48 4) Dispense 20nl of DMSO to columns 1-4. 5) Dispense 1.5ul of 40nM VHR1 in assay buffer to wells in columns 3 through 48. 6) Incubate lidded plate for 1 hour at room temp. 7) Add 3ul of Biomol 8) Incubate for 30 min to allow Biomol signal to develop 9) Read plate on a Perkin Elmer Viewlux at 630nm in absorbance mode Ex1:2 = 630 DF10 Em1:564/250(absorbance); Em2:Clear Light energy = 100000 Measurement time = 5 sec 1x binning Dose-response confirmation 1) Dispense 10ul of assay buffer into columns 1 and 2 of Greiner 384 | |
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