| Assay Method Information | |
| | Inhibition of IDH1 R132H in a Biochemical Assay |
| Description: | IDH1 R132H catalyzes the NADPH-dependent reduction of alpha-ketoglutarate (α-KG) to (2R)-2-hydroxyglutarate (2-HG). NADPH consumption is determined by luminescence.The biochemical reactions were carried out at 32° C. in a 384-well titre plate in a reaction volume of 41 μL in each case and under the following buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used at a final concentration of 1.5 nM. Test compounds were assayed in a concentration range of 0.002 to 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, after which 40 μL of a detection mixture (0.75 μg/ml luciferase, 0.02 U/ml oxidoreductase, 4 μg/mL FMN, 2 μL/ml decanal/ethanol, 50 mM Tris pH 7.5, 0.5% glycerol, 0.01% Tween 20, 0.05% BSA) were added. The luminescence was determined using a luminescence reader (10 seconds measurement time, 1 second integration period, 30% sensitivity). The drop in luminescence is proportional to the activity of mlDH1. IC50 values were determined by interpolation from plots of the relative luminescence against the inhibitor concentration. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |