Amplite® Fluorimetric Monoamine Oxidase Assay Kit *Red Fluorescence*
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
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International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Excitation (nm) | 571 |
Emission (nm) | 584 |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
UNSPSC | 12171501 |
Overview | ![]() ![]() |
Excitation (nm) 571 | Emission (nm) 584 |
Platform
Fluorescence microplate reader
Excitation | 540nm |
Emission | 590nm |
Cutoff | 570nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- MAO standards or test samples (50 µL)
- Add MAO working solution (50 µL)
- Incubate at room temperature for 30-60 min
- Read fluorescence intensity at Ex/Em = 540/590 nm(cut off 570 nm)
Important notes
Thaw all the kit components to room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. Amplite™ Red stock solution (250X):
Add 40 µL of DMSO (Component F) into the vial of Amplite™ Red substrate (Component A). The stock solution should be used promptly. Note: The Amplite™ Red substrate is unstable in the presence of thiols such as dithiothreitol (DTT) and 2-mercaptoethanol. The final concentration of DTT or 2-mercaptoethanol in the reaction should be no higher than 10 µM. The Amplite™ Red substrate is also unstable at high pH (>8.5). Therefore, the reaction should be performed at pH 7 – 8. The provided assay buffer, pH 7.4, is recommended.
2. HRP stock solution (200X):
Add 100 µL of Assay Buffer (Component B) into the vial of horseradish peroxidase (Component C).
3. Plasma Amine Oxidase (PAO) standard solution (20 U/mL):
Add 125 µL of Assay Buffer (Component B) into the vial of Plasma Amine Oxidase Standard (Component E).
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11303
Add 50 µL of 20 U/mL PAO standard solution into 950 µL of Assay Buffer (Component B) to get 1000 mU/mL PAO standard solution (PAO7). Take 1000 mU/mL PAO standard solution and perform 1:3 serial dilutions to get remaining serially diluted PAO standards (PAO6 - PAO1). Note: Higher concentrations of PAO may cause reduced fluorescence signal due to the over oxidation of Amplite™ red substrate (to a non-fluorescent product).
PREPARATION OF WORKING SOLUTION
Add 20 μL of Amplite™ Red stock solution (250X), 25 µL of HRP stock solution (200X) and 25 µL of MAO Substrate (Component D) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.07 mL Monoamine Oxidase (MAO) working solution. Protect from light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of PAO standards and test samples in a solid black 96-well microplate. PAO = plasma amine oxidase standard (PAO1 - PAO7, 1 to 1000 mU/mL); BL = blank control; TS = test sample.
BL | BL | TS | TS |
PAO1 | PAO1 | ... | ... |
PAO2 | PAO2 | ... | ... |
PAO3 | PAO3 | ||
PAO4 | PAO4 | ||
PAO5 | PAO5 | ||
PAO6 | PAO6 | ||
PAO7 | PAO7 |
Table 2. Reagent composition for each well
Well | Volume | Reagent |
PAO1 - PAO7 | 50 µL | serial dilution (1 to 1000 mU/mL) |
BL | 50 µL | Assay Buffer (Component B) |
TS | 50 µL | sample |
- Prepare plasma amine oxidase standards (PAO), blank controls (BL), and test samples (TS) into a 96-well solid black microplate according the the layout provided in Table 1 and Table 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
- Add 50 µL of MAO working solution into each well of the PAO standard, blank control, and test samples to make the total PAO assay volume of 100 µL/well. For a 384-well plate, add 25 µL of MAO working solution into each well instead, for a total volume of 50 µL/well.
- Incubate the reaction for 30 to 60 minutes at room temperature, protected from light.
- Monitor the fluorescence intensity with a fluorescence plate reader at Excitation = 530 - 570, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, cutoff = 570 nm). Note: The contents of the plate can also be transferred to a white clear bottom plate and read by an absorbance microplate reader at the wavelength of 576 ± 5 nm. However, the absorption detection will have a lower sensitivity compared to that of the fluorescence reading.
Product Family
Images
Citations
Authors: Ibero-Baraibar, Idoia
Journal: (2019)
Authors: Ibero-Baraibar, Idoia and Perez-Cornago, Aurora and Ramirez, Maria J and Martínez, J Alfredo and Zulet, M Angeles
Journal: The Journal of nutrition (2016): 897S--904S
Authors: Ibero-Baraibar, Idoia and Perez-Cornago, Aurora and Ramirez, Maria J and Mart{\'\i}nez, J Alfredo and Zulet, M Angeles
Journal: The Journal of nutrition (2015): 897S--904S
References
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