Assay Method Information

Assay Name:  MAO-A/MAO-B Inhibition Assay
Description:  Recombinant human MAO-A and MAO-B (5 mg/mL) were purchased from Sigma-Aldrich, pre-aliquoted and stored at -80 °C. Solutions of test compounds were prepared in DMSO in 2.5 mM for storage and diluted with potassium phosphate buffer (100 mM, pH 7.40, containing KCl 20.2 mM) before use. All the enzymatic reactions were conducted in potassium phosphate buffer (100 mM, pH 7.40, containing KCl 20.2 mM) to a final volume of 500 μL containing kynuramine (45 μM for MAO-A and 30 μM for MAO-B) and various concentrations of test compounds (0-100 μM) with the concentration of DMSO lower than 4%. The reactions were initiated by the addition of MAO-A or MAO-B (7.5 μg/mL) and the solutions were incubated at 37 °C for 30 min. The enzymatic reactions were terminated by the addition of 400 μL NaOH (2 N) and then 1000 μL water, centrifuged for 10 min at 16,000 g. The concentrations of the MAO generated 4-hydroxyquinoline in the reactions were determined by measuring the fluorescence of the supernatant on a Varioskan Flash Multimode Reader (Thermo Scientific) with excitation and emission wavelengths at 310 nm and 400 nm, respectively. A linear calibration curve was constructed by preparing samples containing 4-hydroxyquinoline (0.047-1.56 μM) dissolved in 500 μL potassium phosphate buffer. To each calibration standard, 400 μL NaOH (2 N) and 1000 μL water were added. The appropriate control samples were included to confirm that the test compounds do not fluoresce or quench the fluorescence of 4-hydroxyquinoline under the assay conditions.
Affinity data for this assay
 

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