| Assay Method Information | |
| | Biochemical Assays for Wild-Type IDH1 and IDH2 Enzymes |
| Description: | IDH1 and IDH2 enzymes catalyze the conversion of isocitrate to αKG. Wild-type IDH1 (National Center for Biotechnology Information, Accession: NP_001269316.1) and IDH2 (National Center for Biotechnology Information, Accession: EAX02082.1) proteins containing N-terminal His-tag are expressed in E. coli and purified using nickel affinity chromatography. The enzyme assays are carried out in V-bottom 96 well polypropylene plates containing 100 mM Tris-HCl buffer at pH 7.5, 1 mM DTT, 0.005% TRITON X-100, 120 mM NaCl. For the IDH1 wild-type assay isocitrate, NADP+ and MnCl2 are included at the concentrations of 85 μM, 50 μuM and 20 μM respectively. For the IDH2 wild-type assay isocitrate, NADP+ and MnCl2 are included at the concentrations of 30 μM, 50 μM and 10 μM respectively. Inhibitors dissolved in a DMSO stock solution are diluted in the reaction mixture at a final DMSO concentration of 4%. The enzyme assay is terminated (quenched) by adding ACN (50:50) containing d6-2-ketopentanedioic acid (d6-αKG) as an internal standard for mass spectrometry analysis. Ten microliters of reaction mixture is combined with 100 μL of water, 50 μL of 1 M O-benzylhydroxylamine in pyridine buffer (8.6% pyridine, pH 5), and 50 μL of 1 M N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) in pyridine buffer. Following derivatization at room temperature for one hour, samples are extracted with 600 μL of EtOAc. Four hundred μL of the upper layer is removed, dried under heated nitrogen and reconstituted with 100 μL of MeOH/water (1:1). Ten μL of derivatized sample is injected onto an LC-MS system consisting of a Shimadzu Prominence 20A HPLC system and a The Thermo Quantum Ultra triple quadrupole mass spectrometer. Analytes are separated on a Waters XBridge C18 column (2.1×50 mm, 3.5 μm) with a flow rate of 0.6 mL/minute. Mobile phase A is 0.1% formic acid in water and mobile phase B is MeOH. The αKG signal detected is transformed into analyte concentration using a calibration curve generated using known αKG concentrations. For each compound tested, the % inhibition is calculated using a DMSO control sample as 0% inhibition and a no enzyme control as 100% inhibition. |
| Affinity data for this assay | |
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