| Solution Information | help | |
| Enzyme: | Dual specificity protein phosphatase 3 | |
| inhibitor: | BDBM46223 | |
| 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, 0mM DTT, 0.005% Tween-20 3) 3-O-Methylfluorescein phosphate (OMFP) cyclohexylammonium salt (Sigma-Aldrich, M2629) 4) Upon pre-incubation for 10 min. in the presence of 25 mM DTT, 20 uM VHR1 enzyme stock was aliquoted and flash-frozen. VHR1 working solution at 3 nM in the assay buffer without DTT was prepared using this pre-incubated stock. 4) OMFP working solution: 25 uM OMFP in the assay buffer Procedure for dose-response confirmation assay 1) Using Thermo Scientific MultiDrop Combi dispense 10 uL of OMFP working solution into all columns of black small-volume Greiner plates (784076) 2) Using Thermo Scientific MultiDrop Combi dispense 10 uL of assay buffer in columns 1-2. 3) Using LabCyte Echo 550 add 0.2 uL serially diluted compounds to columns 3-22 and DMSO to columns 1-2 and 23-24. Final concentration of DMSO was 1 % 4) Using Thermo Scientific MultiDrop Comb | |
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