| Assay Method Information | |
| | Biochemical Assay for IDH Inhibition |
| Description: | Solution A was prepared by combining 1 M Tris-HCl pH7.5 (300 μL), 5 M NaCl (450 μL), 1 M MgCl2 (150 μL), 1 M DTT (15 μL), 20 mg/mL BSA (37.5 μL), 20 mM NADPH (20 μL), and 0.5 mM α-KG (40 μL), followed by adding deionized water to a final volume of 15 mL.Reaction buffer was prepared by combining 1 M Tris-HCl pH 7.5 (200 μL), 5 M NaCl (300 μL), 1 M MgCl2 (100 μL), 1 M DTT (10 μL) and 20 mg/mL BSA (25 μL), followed by adding deionized water to a final volume of 10 mL.Solution B was prepared by combining the reaction buffer (5 mL) and 192 M IDH1-R132H (5.21 μL) or 107 μM IDH1-R132C (4.67 μL).148 μL Solution A was added into each sample well of a 96-well plate P1, and then 2 μL of the test compound solution at each concentration and 2 μL of DMSO (negative control/blank) were added into individual sample well respectively. 50 μL of Solution B was added into each of the sample wells having test compounds and one sample well having DMSO only, and 50 μL of reaction buffer was added into another sample well having DMSO only, respectively. P1 was put into BioTek Synergy H4 Microplate reader (BioTek Instruments Inc., Winooski, U.S.), and the program was set as follows: 37° C., shake plate 5 seconds, run kinetic mode, total detection time 20 min, detection intervals 40 seconds, detection mode fluorescence (at Excitation 340 nm, Emission 460 nm). The data were exported, the relative enzyme activity and the half-maximal inhibitory concentration (IC50) for the test compound were calculated. Relative enzyme activity was calculated as the absolute value of the slope of the Emission 460 reads of each well plotted against time (which represents the conversion rate of ca-KG to 2-HG through IDH1 modulated utilization of NADPH). The Dose-response curve was drawn and the IC50 was calculated via fitting. The IDH inhibition and selectivity of each test compound can be evaluated according to the effect of the compounds on the IDH enzyme activity. |
| Affinity data for this assay | |
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