| Solution Information | help | |
| Enzyme: | Diphosphomevalonate decarboxylase | |
| inhibitor: | BDBM38345 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Diphosphomevalonate Decarboxylase Protocol for 384-well HTS Purified recombinant MK enzyme was provided by Dr. Thomas Leyh, Albert Einstein College of Medicine of Yeshiva University. Final Assay concentrations: 25 uL/well 50 mM HEPES buffer, pH 8.0 50 mM KCl 4 mM MgCl2 0.4 mM ATP 5uM diphosphomevalonate (DPM) 2 mM Potassium Phosphoenolpyruvate 20 uM NADH 3 units/ml of Rabbit muscle lactic dehydrogenase (LDH) (Sigma) 3 units/ml of Rabbit muscle pyruvate kinase (Sigma) 30 nM of DPM-DC 4% DMSO Full reaction conditions are listed above. Background conditions are the same as above except that the substrate (DPM) is left out. The assay was run in 384-well black opque plates. Fifteen ul substrate/background was added to plates using the BeckmanCoulter Biomek FX liquid handling workstation. Compounds were then added in 1 ul volumes using the FX. The reaction was initiated by the addition of 10 ul of DPM-DC using the Titertek Multidrop peristaltic pipetting platform. The test p | |
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