Signaling Pathway Assays
Monoamine Oxidase Inhibitor Screening Kit (BA0188)
- SKU:
- BA0188
- Product Type:
- Assay
- Instrument:
- Microplate Reader
- Sample Type:
- Monoaime Oxidase
- Research Area:
- Oxidative Stress
- Research Area:
- Reactive Oxygen Species
- Research Area:
- High-Throughput Screening
Description
Monoamine Oxidase Inhibitor Screening Kit (BA0188) (BA0188)
The Monoamine Oxidase Inhibitor Screening Kit (SKU: BA0188) provides a convenient fluorimetric means to screen for inhibitors of monoamine oxidase (MAO) enzymes. Monoamine oxidases are a family of mitochondrial enzymes that catalyse the oxidative deamination of monoamines, existing as two isoforms, MAO-A and MAO-B, with differing inhibitor selectivity and tissue distribution; MAO dysfunction is implicated in a number of neurological disorders and MAO inhibitors are among the major classes of drug prescribed for depression, Parkinson's and Alzheimer's diseases. In this assay MAO reacts with p-tyramine, a substrate for both MAO-A and MAO-B, resulting in the formation of hydrogen peroxide, which is determined by a fluorimetric method at 530/585 nm. The assay is simple, sensitive, stable and adaptable to high-throughput screening. It is intended for research use only.
| Product Name: | Monoamine Oxidase Inhibitor Screening Kit (BA0188) |
| SKU: | BA0188 |
| Detection Method: | Fluorimetric (lambda ex/em = 530/585 nm) |
| Sample Type: | Purified MAO-A and MAO-B with test compounds |
| Species Reactivity: | All |
| Assay Time: | Approximately 35 min plus incubations |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20C |
| Shelf Life: | 6 months after receipt |
| Shipping: | Gel Pack |
A fluorimetric screening assay for the identification and evaluation of monoamine oxidase inhibitors. MAO reacts with p-tyramine to form hydrogen peroxide, which is detected fluorimetrically at 530/585 nm. The homogeneous mix-incubate-measure format is stable and adaptable to high-throughput screening. Neither MAO-A nor MAO-B enzyme is included in the kit.
- Safe, non-radioactive assay
- Homogeneous and convenient mix-incubate-measure format with no wash or reagent-transfer steps
- Robust and amenable to high-throughput screening on liquid-handling systems
- High-throughput screening for MAO inhibitors
- Evaluation of MAO inhibitor potency and selectivity
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 | Dilute purified MAO-A to 3 U/mL and MAO-B to 6 U/mL using dH2O, and dissolve the test compounds in the solvent of choice (DMSO concentration should be 10 v/v% or less in the 5 uL added when screening with human MAO). For human MAO-A, prepare a 1.5-fold dilution of p-Tyramine; for human MAO-B, a 4-fold dilution. |
| 2 | Transfer 45 uL of either diluted MAO-A or MAO-B into separate wells, reserving at least one well for no substrate (Blank) and one without inhibitor (No Inhibitor Control). |
| 3 | Add 5 uL of solvent to the No Inhibitor Control and Blank wells; add 5 uL of test compound or positive inhibitor control to the remaining MAO wells. Mix and incubate for 15 minutes at 25C. |
| 4 | Prepare Working Reagent by mixing, per well: 50 uL Assay Buffer, 1 uL diluted p-Tyramine, 1 uL Dye Reagent and 1 uL HRP Enzyme. Prepare Blank Working Reagent by substituting 1 uL dH2O for the p-Tyramine. Transfer 50 uL Blank Working Reagent to the Blank wells and 50 uL Working Reagent to the remaining wells, and briefly tap to mix. |
| 5 | Incubate for 20 minutes in the dark and read fluorescence intensity at lambda exc = 530 nm and lambda em = 585 nm. |
% Activity = [(RFU_TestCpd - RFU_Blank) / (RFU_NoInhibitor - RFU_Blank)] x 100%, where RFU_TestCpd, RFU_Blank and RFU_NoInhibitor are the values for the test compound, the Blank (no substrate) and the No Inhibitor wells respectively.
| Component | Quantity | Storage |
| Assay Buffer (pH 7.4) | 12 mL | -20C |
| p-Tyramine | 120 uL | -20C |
| Pargyline (20 mM) | 50 uL | -20C |
| HRP Enzyme | 120 uL | -20C |
| Clorgyline (20 mM) | 50 uL | -20C |
| Dye Reagent | 120 uL | -20C |
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