Monoamine Oxidase Assay Kit, BioAssay(TM)

Monoamine Oxidases (MAO, EC 1.4.3.4) are a family of mitochondrial enzymes that catalyze the oxidative deamination of monoamines. MAO dysfunction is thought to be responsible for a number of neurological disorders. Unusually high or low levels of MAOs in the body have been associated with depression, schizophrenia, substance abuse, attention deficit disorder, migraines, and irregular sexual maturation. MAO inhibitors are one of the major classes of drug prescribed for the treatment of depression.\nMAO Assay Kit provides a convenient fluorimetric means to measure MAO enzyme activity. In the assay, MAO reacts with p-tyramine, a substrate for both MAO-A and MAO-B, resulting in the formation of H2O2, which is determined by a fluorimetric method (lem/ex=585/530nm). The assay is simple, sensitive, stable and high- throughput adaptable.\n\nKey Features:\nSafe. Non-radioactive assay.\nSensitive and accurate. As low as 0.01 U/L MAO activity can be quantified.\nHomogeneous and convenient. "Mix-incubate-measure" type assay.\nNo wash and reagent transfer steps are involved.\nRobust and amenable to HTS: can be readily automated on HTS liquid handling systems for processing thousands of samples per day.\n\nApplications:\nMAO-A/B activity determination in biological samples.\nEvaluation and screening for MAO inhibitors.\n\nKit Contents:\nAssay Buffer: 12ml (pH 7.4) p-Tyramine: 300ul\nPargyline: 50ul 20mM HRP Enzyme: 120ul\nClorgyline: 50ul 20mM Dye Reagent: 120ul\nHydrogen Peroxide: 100ul 3% H2O2\nStorage conditions: store Assay Buffer and Hydrogen Peroxide at 4 degrees C and other reagents at -20 degrees C. This product is shipped on ice. Shelf life: 6 months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nAssay Procedure:\nNote: (1) thiols (b-mercaptoethanol, dithioerythritol etc) at > 10uM interfere with this assay and should be avoided in sample preparation.\n(2). Samples should be free of particle or precipitates. MAO can be extracted from a tissue by homogenization and differential centrifugation, e.g. Biochem. J. (1968) 108: 95. Store sample at -80 degrees C.\n(3). Prior to assay, concentrations of protein, inhibitor, substrate and incubation time may need to be established for a given sample. Use black flat-bottom plates. Prior to assay, bring all components to room temperature, briefly centrifuge tubes before opening. Dilute the 20mM inhibitors with H2O to 10uM (e.g. mix 5ul 20mM inhibitor with 10ml H2O).\n1. To determine MAO-A activity, use 1mM p-tyramine substrate and include a control with 0.5uM MAO-A inhibitor clorgyline. Samples: dilute sample in Assay Buffer. Transfer 45ul of each sample into two separate wells. Add 5ul H2O (SAMPLE) and 5ul 10uM clorgyline (CONTROL). Mix and incubate for 10 min at room temperature for the inhibitor to block MAO-A activity.\n2. Calibrator. Mix 5ul H2O2 with 1555ul H2O. Further dilute 5ul of the resulting H2O2 in 780ul H2O to give 20uM H2O2. Dilute calibrator with H2O to give 20, 10, 5 and 0uM H2O2. Transfer 50ul calibrators into separate wells of the assay plate.\n3. Prepare enough Working Reagent for all sample and calibrator wells. For each well, mix: 50ul Assay Buffer, 1ul p-tyramine, 1ul Dye Reagent and 1ul HRP Enzyme. Transfer 50ul Working Reagent to all wells. Briefly tap plate to mix.\n4. Incubate for 20 min in the dark. Read fluorescence intensity at lexc=530nm and lem=585nm. To measure MAO-B activity, use 1mM p-tyramine and include a control with 0.5uM pargyline (MAO-B inhibitor). Procedure is the same as for MAO-A determination. To screen for MAO inhibitors or characterize inhibitor potency (IC50), mix 5ul inhibitor with 45ul sample and incubate for at least 10 min to allow the inhibitor to interact with the enzyme, prior to adding the Working Reagent.\n\nCalculation:\nPlot H2O2 calibration curve and determine its Slope (uM-1). MAO enzyme activity in the sample is calculated as\nMAO Activity =\nRFUSAMPLE

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SPECIFICATIONS

Catalog Number

M4450-05

Size

1Kit

References

1. Ivanovic, I.D. and Majkic-Singh, N. (1988). Determination of platelet monoamine oxidase by new continuous spectrophotometric method. J Clin Chem Clin Biochem. 26: 447-51.\n2. Suzuki, O. et al. (1976). A simple fluorometric assay for type B monoamine oxidase activity in rat tissues. J. Biochem. 79: 1297-1299.\n3. Youdim, M. B. H. & Tenne, M. (1987). Assay and purification of liver monoamine oxidase. Methods Enzymol. 142: 617-626.