| Assay Method Information | |
| | IDH1 Biochemical Assay |
| Description: | The activity of IDH1 R132H and IDH1 R132C was measured in 384-well plates by coupling NADPH consumption to a diaphorase/resazurin-based detection system and measuring resorufin production (Ex544/Em590) as described previously. For testing the potency of small molecule inhibitors, an end point-coupled assay system was used, with 2-4 nM enzyme. In this assay, the reactions were run in the α-KG to 2-HG direction with the concomitant oxidation of NADPH to NADP. For IDH1 R132H, a final concentration of 1 mM α-KG and 4 μM NADPH was used; for IDH1 R132C, a final concentration of 200 μM α-KG and 4 μM NADPH was used. The NADPH remaining at the end of a 1-h incubation reaction was measured by the addition of 20 μM resazurin and 10 μg/ml diaphorase, which facilitated the stoichiometric conversion of resazurin to the highly fluorescent resorufin. To test the reversibility of the binding of compound to enzyme, 10× IC50 of the compound and 100× enzyme were incubated for 1 h, at which point the sample was diluted 100-fold and the enzyme reaction was run in kinetic mode monitoring the consumption of NADPH at 340 nm. |
| Affinity data for this assay | |
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