Description
Monoamine Oxidase Assay Kit (BA0130) (BA0130)
This Monoamine Oxidase Assay Kit (SKU: BA0130) provides a convenient fluorimetric means of measuring monoamine oxidase (MAO) enzyme activity in biological samples. Monoamine oxidases (EC 1.4.3.4) are mitochondrial enzymes that catalyse the oxidative deamination of monoamines, and their dysfunction has been associated with a range of neurological disorders including depression, schizophrenia and migraine; MAO inhibitors are a major class of antidepressant drug. In this assay MAO reacts with p-tyramine, a substrate for both MAO-A and MAO-B, to form hydrogen peroxide, which is measured fluorimetrically at λex/em = 530/585 nm. The assay is simple, sensitive, stable and adaptable to high-throughput screening, and allows determination of MAO-A and MAO-B activity separately through the use of selective inhibitors.
| Product Name: | Monoamine Oxidase Assay Kit (BA0130) |
| SKU: | BA0130 |
| Detection Method: | Fluorimetric (λex/em = 530/585 nm), H2O2 detection |
| Detection Range: | As low as 0.01 U/L MAO activity |
| Sample Type: | Biological samples (tissue extracts) |
| Species Reactivity: | All |
| Assay Time: | 20 minutes (plus 10-minute inhibitor pre-incubation) |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | Store all components at -20°C. |
| Shelf Life: | 6 months after receipt. |
| Shipping: | Gel Pack |
MAO reacts with p-tyramine to produce hydrogen peroxide, which is detected by a horseradish peroxidase-coupled fluorimetric reaction at λex/em = 530/585 nm. Selective inhibitors (clorgyline for MAO-A and pargyline for MAO-B) allow the two activities to be distinguished. One unit of MAO catalyses the formation of 1 µmole of H2O2 per minute under the assay conditions.
- Safe, non-radioactive assay.
- Sensitive and accurate, quantifying MAO activity as low as 0.01 U/L.
- Homogeneous and convenient 'mix-incubate-measure' assay with no wash or reagent transfer steps.
- Robust and amenable to high-throughput screening, readily automated for thousands of samples per day.
- MAO-A and MAO-B activity determination in biological samples.
- Evaluation and screening for MAO inhibitors.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Notes: thiols (β-mercaptoethanol, dithioerythritol, etc.) above 10 µM interfere and should be avoided; samples should be free of particles or precipitates and MAO can be extracted from tissue by homogenisation and differential centrifugation, with samples stored at -80°C; protein, inhibitor and substrate concentrations and incubation time may need to be established for a given sample. |
| 2 | Use black flat-bottom plates, bring all components to room temperature and briefly centrifuge tubes before opening; dilute the 20 mM inhibitors with water to 10 µM (e.g. 5 µL of 20 mM inhibitor in 10 mL water). |
| 3 | For MAO-A activity, use 1 mM p-tyramine substrate and include a control containing 0.5 µM of the MAO-A inhibitor clorgyline; dilute the sample in Assay Buffer, transfer 45 µL of each sample into two wells and add 5 µL water (sample) and 5 µL of 10 µM clorgyline (control), then mix and incubate 10 minutes at room temperature to block MAO-A activity. |
| 4 | Calibrator: mix 5 µL H2O2 with 1400 µL water, further dilute 5 µL of the result in 780 µL water to give 20 µM H2O2, and dilute to 20, 10, 5 and 0 µM; transfer 50 µL of each calibrator into separate wells. |
| 5 | Prepare sufficient Working Reagent by mixing, per well, 50 µL Assay Buffer, 1 µL p-tyramine, 1 µL Dye Reagent and 1 µL HRP Enzyme, and transfer 50 µL Working Reagent to all wells, briefly tapping to mix. |
| 6 | Incubate 20 minutes in the dark and read the fluorescence intensity at λex = 530 nm and λem = 585 nm. |
| 7 | To measure MAO-B activity, use 1 mM p-tyramine and include a control with 0.5 µM pargyline, following the same procedure; to screen for inhibitors or determine IC50, mix 5 µL inhibitor with 45 µL sample and pre-incubate at least 10 minutes before adding the Working Reagent. |
Plot the H2O2 calibration curve and determine its slope (µM^-1). MAO Activity (U/L) = (RFU_SAMPLE - RFU_CONTROL) / (Slope x t), where RFU_SAMPLE and RFU_CONTROL are the fluorescence values of the sample and the sample control (in the presence of the relevant inhibitor, pargyline or clorgyline) and t is the incubation time (20 min). One unit of MAO catalyses the formation of 1 µmole of H2O2 per minute under the assay conditions.
| Component | Quantity | Storage |
| Assay Buffer (pH 7.4) | 12 mL | -20°C |
| p-Tyramine | 120 µL | -20°C |
| Pargyline (20 mM) | 50 µL | -20°C |
| HRP Enzyme | 120 µL | -20°C |
| Clorgyline (20 mM) | 50 µL | -20°C |
| Dye Reagent | 120 µL | -20°C |
| Hydrogen Peroxide (3% H2O2) | 100 µL | -20°C |
