CHEMBL5733209

Fluorescent-based In Vitro Assa: The assay was performed in Optiplate 96-wells black plates, with each reaction well containing a mixture of 25 mM sodium acetate buffer pH 4.5 and a fixed amount of protein (2 μg) in a volume of 85 μL. After 30 mins of pre-incubation with test compounds (diluted 20× from DMSO stock solutions at different concentrations), the fluorogenic probe was added (diluted 20× from EtOH stock solution, final concentration 5 μM). After incubation for 3 hours at 37° C., the reactions were stopped with 50 μL of methanol and 100 μL of a 2.5 mg/mL NalO4 fresh solution in 100 mM Glycine/NaOH pH 10.6. The plate was incubated at 37° C. for 2 hours in the dark and fluorescence intensities were measured at excitation/emission wavelengths of 360/446 nm. Negative control samples consisted of the same incubation mixture in the absence of protein extracts.