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| Solution Information | help | |
| Enzyme: | Dual specificity protein phosphatase 3 | |
| inhibitor: | BDBM46222 | |
| 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) 3-O-Methylfluorescein phosphate (OMFP) cyclohexylammonium salt (Sigma-Aldrich, M2629) 4) VHR1 working solution: 3 nM VHR1 solution in the assay buffer 4) OMFP working solution: 25 uM OMFP in the assay buffer Procedure for dose-response confirmation assay 1) Dispense 10 ul of OMFP working solution into all columns of black small-volume Greiner plates (784076) 2) Dispense 10 uL of assay buffer in columns 1-2. 3) Add serially diluted compounds to columns 3-22 and DMSO to columns 1-2 and 23-24. Final concentration of DMSO was 1.575% 4) Dispense 10 of VHR1 working solution to wells in columns 3 through 24. 5) Incubate lidded plate for 1 hour at room temp. 6) Read plate on M5 plate reader in fluorescence mode (ex: 480nm; 3m: 520nm; dichroic mirror is 515 nm) | |
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