Table 2Diaphorase/resazurin-coupled IDH1 R132H secondary assay protocol

1Compound1 μLCompound or DMSO
2Reagent25 μLNADPH and α-KG buffer (0.004 mM and 1 mM final conc., resp.) or buffer-only control
3Spin1 minCentrifuge at 271xg
4Reagent25 μLIDH1 R132H or no enzyme buffer (2.6 and 0 nM final conc., resp.)
5Spin1 minCentrifuge at 271xg
6Shake1 minShake on orbital shaker
7Time50 minRT incubation
8Reagent25 μLDiaphorase and resazurin buffer (0.32 μM and 10 μM final conc., resp.)
9Spin1 minCentrifuge at 271xg
10Shake1 minShake on orbital shaker
11DetectorFluorescenceSpectramax M5; EX 544, EM 590, 590 cut-off filter; PMT auto, medium precision

From: ML309: A potent inhibitor of R132H mutant IDH1 capable of reducing 2-hydroxyglutarate production in U87 MG glioblastoma cells

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