| Assay Method Information | |
| | Inhibition Assay |
| Description: | The DAAO inhibitory activity was measured by assaying the amount of hydrogen peroxide (H2O2) produced by reacting DAAO protein with flavin adenine dinucleotide (FAD) and D-alanine. The amount of H2O2 was determined by measuring the fluorescence generated on conversion of Amplex red (manufactured by Invitrogen Co.) into resorufin by the reaction of H2O2 with horseradish peroxidase (HRP). 4 uL of 4% dimethyl sulfoxide (DMSO) buffer (50 mM sodium phosphate (pH 7.5), 0.02% CHAPS) solution of the test compound was added to 384-well black, low volume plate, a mixed solution (4 uL) of recombinant human DAAO protein (15 nM), which had been expressed in Escherichia coli and purified, and 18 uM FAD was added, and the mixture was incubated at room temperature for 15 min. After incubation, a mixed buffer (4 uL) of 2.25 mM D-alanine, 1.5 U/mL HRP and 150 uM Amplex red was added, the mixture was incubated at room temperature for 30 min. |
| Affinity data for this assay | |
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