Monoamine Oxidase Assay Kit

Catalog No. ABIN1000321

Product Details

Target: MAO (Monoamine Oxidase (MAO))

Application: Activity Assay (AcA)

Specificity: 0.01 U/L

Characteristics: Safe. Non-radioactive assay.
Sensitive and accurate. As low as 0.01 U/L MAO activity can be quantified.
Homogeneous and convenient. Mix-incubate-measure type assay. No wash and reagent transfer steps are involved.
Robust and amenable to HTS: can be readily automated on HTS liquid handling systems for processing thousands of samples per day.

Components: Assay Buffer: 12 mL (pH 7.4). p-Tyramine: 300 µL. Pargyline: 50 µL 20 mM. HRP Enzyme: 120 µL. Clorgyline: 50 µL 20 mM. Dye Reagent: 120 µL. Hydrogen Peroxide: 100 µL 3% H2O2.

Material not included: Pipetting devices, centrifuge tubes, black flat bottom 96-well plate (e.g. Corning Costar).

Application Details

Application Notes: MAO-A/B activity determination in biological samples. Evaluation and screening for MAO inhibitors.

Protocol: Note: (1) thiols (beta-mercaptoethanol, dithioerythritol etc) at > 10 µMinterfere with this assay and should be avoided in sample preparation. (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°C. (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 20 mM inhibitors with H2O to 10 µM(e.g. mix 5 µL 20 mM inhibitor with 10 mL H2O).
1. To determine MAO-A activity, use 1 mM p-tyramine substrate and include a control with 0.5 µMMAO-A inhibitor clorgyline. Samples: dilute sample in Assay Buffer. Transfer 45 µL of each sample into two separate wells. Add 5 µL H2O (SAMPLE) and 5 µL 10 µMclorgyline (CONTROL). Mix and incubate for 10 min at room temperature for the inhibitor to block MAO-A activity.
2. Calibrator. Mix 5 µL H2O2 with 1555 µL H2O. Further dilute 5 µL of the resulting H2O2 in 780 µL H2O to give 20 µMH2O2. Dilute calibrator with H2O to give 20, 10, 5 and 0 µMH2O2. Transfer 50 µL calibrators into separate wells of the assay plate.
3. Prepare enough Working Reagent for all sample and calibrator wells. For each well, mix: 50 µL Assay Buffer, 1 µL p-tyramine, 1 µL Dye Reagent and 1 µL HRP Enzyme. Transfer 50 µL Working Reagent to all wells. Briefly tap plate to mix.
4. Incubate for 20 min in the dark. Read fluorescence intensity at exc = 530nm and em = 585nm. To measure MAO-B activity, use 1 mM p-tyramine and include a control with 0.5 µMpargyline (MAO-B inhibitor). Procedure is the same as for MAO-A determination. To screen for MAO inhibitors or characterize inhibitor potency (IC50), mix 5 µL inhibitor with 45 µL sample and incubate for at least 10 min to allow the inhibitor to interact with the enzyme, prior to adding the Working Reagent.

Restrictions: For Research Use only

Handling

Storage: 4 °C

Target Details

Target: MAO (Monoamine Oxidase (MAO))

Alternative Name: Monoamine Oxidase (MAO Products)

Synonyms: Z-MAO Kit, maob Kit, moa Kit, wu:fb68b05 Kit, wu:fo76d11 Kit, wu:fq38g06 Kit, zgc:85761 Kit, mao Kit, Maob Kit, monoamine oxidase Kit, mao Kit, azo2784 Kit, AZL_b03560 Kit, Psyc_0841 Kit

Background: Rapid, quantitative, fluorimetric (530nm/585nm) determination of monoamine oxidase activity or MAO inhibitor screen.
Procedure: 30 min.

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. This MAO 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 (gamma em/ex = 585/530nm). The assay is simple, sensitive, stable and high-throughput adaptable.