| Assay Method Information | |
| | RapidFire-MS/MS and NADPH Assays |
| Description: | RapidFire-MS/MS measurements of mutant IDH1 and IDH2 reactions were conducted at room temperature in 384-well Greiner polypropylene microtiter plates in a total volume of 20 μL of assay buffer. Final compound concentrations were typically varied from 0.5 to 100,000 nM, though in the case of tight-binding inhibition a range of 0.4 nM to 2 μM was used with a 1.5-fold dilution scheme. The αKG concentration was fixed at 500 μM, the NADPH concentration was fixed at 50 μM, and mutant IDH1 and IDH2 were fixed at 15 nM. Reactions were conducted in duplicate and quenched with final concentrations of 100 mM EDTA. After each addition plates were centrifuged for 60 s to ensure complete mixing of reagents. Endpoint data was obtained after 2 h for R132C IDH1, R132G IDH1, R140Q IDH2 and R172S IDH2 and after 4 h for R132H IDH1. For the RapidFire-MS/MS analysis, a 0.5 μL aliquot of each well was diluted 200-fold into a separate analysis plate containing 100 μL of a f |
| Affinity data for this assay | |
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