Table 4Diaphorase/resazurin-coupled WT IDH1 secondary assay protocol

SequenceParameterValueDescription
1Compound1 μLCompound Library
2Reagent40 μLWT IDH1 or no enzyme buffer (0.65 & 0 nM final conc., resp.)
3Time60 minRT incubation
4Reagent10 μLNADP+, diaphorase, resazurin, isocitrate buffer (0.05, 0.00053, 0.04 & 0.07 mM final conc., resp.)
5Time30 minRT incubation
6Reagent25 μLStop buffer (2% SDS final concentration)
7Shake1 minShake on orbital shaker
8DetectorFluorescenceSpectramax M5 in endpoint mode: 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

Cover of Probe Reports from the NIH Molecular Libraries Program
Probe Reports from the NIH Molecular Libraries Program [Internet].

NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.