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Amplite® Fluorimetric Malondialdehyde (MDA) Quantitation Kit *Enhanced Selectivity*
Example protocol
AT A GLANCE
Protocol summary
- Prepare and add MDA standards or test samples (50 uL)
- Prepare and add MDA Green™ working solution (50 uL)
- Incubate at room temperature for 6 minutes
- Add MDA Stopping Solution (50 uL)
- Monitor fluorescence intensity at Ex/Em=480/555 nm
Important notes
Thaw all the kit components to room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
1. MDA standard solution (100 mM):
Add 100 uL of ddH2O into the vial of MDA Standard (Component C) to make 100 mM MDA standard solution.
2. MDA Green™ stock solution (250 X):
Add 20 uL of DMSO (Component E) into one vial of MDA Green™ (Component A) to make vial of MDA Green™ stock solution. Note: All stock solutions should be stored at -20oC after preparation. Avoid repeated freeze-thaw cycles.
PREPARATION OF STANDARD SOLUTION
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/10072
Add 10 uL of 100 mM MDA standard solution into 990 uL of MDA Assay Buffer (Component B) to generate 1000 uM MDA standard solution (MDA1). Take 1000 uM MDA standard solution (MDA1) and perform 1:3 serial dilutions to get serially diluted MDA standards (MDA2 - MDA7) with MDA Assay Buffer (Component B).
PREPARATION OF WORKING SOLUTION
MDA Green™ working solution:
Add 20 uL of 250 X MDA Green™ stock solution into 5 mL of MDA Assay Buffer (Component B), and mix well to make MDA Green™ working solution. Note: This MDA Green™ working solution should be prepared before the experiment, and kept from light.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of MDA standards and test samples in a solid black 96-well microplate. MDA= MDA Standards (MDA1 - MDA7, 1000 to 1.37 µM), BL=Blank Control, TS=Test Samples.
| BL | BL | TS | TS |
| MDA1 | MDA1 | ... | ... |
| MDA2 | MDA2 | ... | ... |
| MDA3 | MDA3 | ||
| MDA4 | MDA4 | ||
| MDA5 | MDA5 | ||
| MDA6 | MDA6 | ||
| MDA7 | MDA7 |
Table 2. Reagent composition for each well.
| Well | Volume | Reagent |
| MDA1 - MDA7 | 50 µL | Serial Dilutions (1000 to 1.37 µM) |
| BL | 50 µL | MDA Assay Buffer (Component B) |
| TS | 50 µL | test sample |
- Prepare MDA standards (MDA), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 25 uL of reagent per well instead of 50 uL.
- Add 50 uL of MDA Green™ working solution to each well of MDA standard, blank control, and test samples. For a 384-well plate, add 25 uL of MDA Green™ working solution into each well instead.
- Incubate the reaction at room temperature for 5-6 minutes, protected from light.
- Add 50 uL of MDA Stopping Solution (Component D) to each well of the reaction. For a 384-well plate, add 25 uL of MDA Stopping Solution (Component D) into each well instead.
- Monitor the fluorescence intensity with a fluorescence plate reader at Ex/Em=480/555 nm (Cutoff= 530 nm).
Citations
Authors: Bakirezer, S. D., Yaltirik, C. K., Kaya, A. H., Yilmaz, S. G., Ozdogan, S., Billur, D., Isbir, T.
Journal: In Vivo (2019): 811-814
Authors: Sharif Usman, S., Dahiru, M., Abdullahi, B., Abdullahi, S. B., Maigari, U. M., Ibrahim Uba, A.
Journal: Heliyon (2019): e02214
Authors: Polzin, A., Dannenberg, L., Schneider, T., Knoop, B., Naguib, D., Helten, C., Pohl, M., Kelm, M., Zeus, T., Hohlfeld, T.
Journal: Pharmacology (2019): 23-29
Authors: Khedr, M. A., El-Araby, H. A., Konsowa, H. A., Sokar, S. S., Mahmoud, M. F., Adawy, N. M., Zakaria, H. M.
Journal: Clin Exp Hepatol (2019): 81-87
Authors: Islam, M. R., Islam, M. R., Ahmed, I., Moktadir, A. A., Nahar, Z., Islam, M. S., Shahid, S. F. B., Islam, S. N., Islam, M. S., Hasnat, A.
Journal: SAGE Open Med (2018): 2050312118773953
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