Table 8Stepwise protocol of the rGAPDH counterscreen in 1536-well format

SequenceParameterValueDescription
1Reagent3 μLSubstrate buffer (final conc: 2 mM G3P, 1 mM NAD+, 0.1 mM resazurin, 0.1 mg/mL diaphorase)
2Compound23 nLCompound Library
3Reagent1 μLrGAPDH or 0× control (final conc: 2 nM and buffer only, resp.)
4DetectorFluorescenceInitial read (ViewLux; ex 525, em 598, 1 second exposure, Bodipy Mirror)
4Time30 minRT incubation
5DetectorFluorescenceFinal read (ViewLux; ex 525, em 598, 1 second exposure, Bodipy Mirror)

From: Discovery of two small molecule inhibitors, ML387 and ML388, of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

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