Amplite® Fluorimetric Myeloperoxidase 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 |
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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 | SDSProtocol |
Excitation (nm) 571 | Emission (nm) 584 |
Platform
Fluorescence microplate reader
Excitation | 540 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Example protocol
AT A GLANCE
Protocol summary
- MPO standards or test samples (50 µL)
- Add MPO 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 E) 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. H2O2 stock solution (500X, 10 mM):
Add 10 µL of 3% H2O2 (0.88M, Component C) into 870 µL of Assay Buffer (Component B). Note: The diluted H2O2 solution is not stable. The unused portion should be discarded.
3. Myeloperoxidase (MPO) standard solution (200 mU/mL):
Add 50 µL of Assay Buffer (Component B) into the vial of Myeloperoxidase Standard (Component D). Note: One vial contains approximately 5 - 10 mU myeloperoxidase.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11301
Add 20 µL of 200 mU/mL MPO standard solution into 380 µL of Assay Buffer (Component B) to get 10 mU/mL MPO standard solution (MPO7). Take 10 mU/mL MPO standard solution to perform 1:3 serial dilutions to get remaining serially diluted MPO standards (MPO6 - MPO1).
PREPARATION OF WORKING SOLUTION
Add 20 μL of Amplite™ Red Stock Solution (250X) and 10 μL of H2O2 (500X) into 5 mL of Assay Buffer (Component B) to make a total volume of 5.03 mL MPO working solution. Protect from light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of MPO standards and test samples in a 96-well solid black microplate. MPO= myeloperoxidase standards (MPO1 - MPO7, 0.01 to 10 mU/mL); BL=blank control; TS = test samples.
BL | BL | TS | TS |
MPO1 | MPO1 | ... | ... |
MPO2 | MPO2 | ... | ... |
MPO3 | MPO3 | ||
MPO4 | MPO4 | ||
MPO5 | MPO5 | ||
MPO6 | MPO6 | ||
MPO7 | MPO7 |
Table 2. Reagent composition for each well. Note that high concentration of MPO may cause reduced fluorescence signal due to the over oxidation of Amplite™ Red substrate (to a non-fluorescent product).
Well | Volume | Reagent |
MPO1 - MPO7 | 50 µL | Serial Dilution (0.01 to 10 mU/mL) |
BL | 50 µL | Assay Buffer (Component B) |
TS | 50 µL | test sample |
- Prepare myeloperoxidase standards (MPO), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 µL of reagent per well instead of 50 µL.
- Add 50 µL of MPO working solution to each well of myeloperoxidase standard, blank control, and test samples to make the total MPO assay volume of 100 µL/well. For a 384-well plate, add 25 µL of MPO 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 nm, Emission = 590 - 600 nm (optimal Ex/Em = 540/590 nm, cut off = 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. The absorption detection has lower sensitivity compared to that of the fluorescence reading.
Images
Citations
Authors: Akentieva, Natalia and Sanina, Natalia and Gizatullin, Artur and Shkondina, Natalia and Andreeva, Anna and Shram, Stanislav and Aldoshin, Sergei
Journal: (2022)
References
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