Amplite® Colorimetric Malondialdehyde (MDA) Quantitation Kit
Malondialdehyde (MDA) is natural byproduct of lipid peroxidation and is widely used as a key indicator to determine the oxidative stress and free radical formation. Measurement of MDA has historically relied on the reaction with thiobarbituric acid (TBA) to results in a compound that can be measured colorimetrically at or fluorimetrically. But this assay is not specific to MDA and also takes place under acidic conditions at 90-100 °C. There have been a number of commercial ELISA kits, which makes it more expensive and tedious. The Amplite® Colorimetric Malondialdehyde (MDA) Quantitation Kit offers the most rapid and convenient method to measure MDA without the TBARS heating steps. MDA Blue™ reacts with MDA to generate a blue color product which is measured at 695 nm with absorbance microplate reader. This assay is very fast and specific to MDA with little interference from other aldehydes.
Example protocol
AT A GLANCE
Protocol summary
- Prepare test samples along with serially diluted MDA standards (50 µL)
- Add MDA Blue™ stock solution (10 µL)
- Incubate at room temperature for 10 - 30 minutes
- Add Reaction Solution (40 µL)
- Monitor OD increase at 695 nm
Important notes
Thaw all the components at room temperature before starting the experiment.
PREPARATION OF STOCK SOLUTION
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
1. MDA standard solution (100 mM):
Add 100 µL of ddH2O into MDA Standard vial (Component C) to make 100 mM MDA stock solution.
PREPARATION OF STANDARD SOLUTION
MDA standard
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/10070
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/10070
Add 4 µL of 100 mM MDA standard into 996 µL of Dilution Buffer (Component B) to get 400 µM MDA solution (MDA7). Then perform 1:2 serial dilutions in dilution buffer to get serially diluted MDA standards (MDA6 - MDA1).
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of MDA standards and test samples in a 96-well clear bottom microplate. MDA= MDA Standard (MDA1 - MDA7, 6.25 to 400 µM), BL=Blank Control, TS=Test Sample.
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 Dilution (6.25 to 400 µM) |
BL | 50 µL | Dilution 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 µL of reagent per well instead of 50 µL.
- Add 10 µL MDA Blue™ (Component A) solution into each well of MDA standard, blank control, and test samples. For a 384-well plate, use 5 µL MDA Blue™ solution into each well.
- Incubate the reaction mixture at room temperature for 10 - 30 minutes.
- Add 40 µL of Reaction Solution (Component D) to make the total assay volume of 100 µL/well. For a 384-well plate, use 20 µL of Reaction Solution, for a total assay volume of 50 µL/well.
- Incubate the final reaction mixture at room temperature for 30 - 60 minutes.
- Monitor absorbance increase with an absorbance plate reader with path-check correction at OD of 695 ~ 700 nm.
Citations
View all 2 citations: Citation Explorer
Hydrogen-generating Si-based agent protects against skin flap ischemia--reperfusion injury in rats
Authors: Otani, Naoya and Tomita, Koichi and Kobayashi, Yuki and Kuroda, Kazuya and Koyama, Yoshihisa and Kobayashi, Hikaru and Kubo, Tateki
Journal: Scientific reports (2022): 1--8
Authors: Otani, Naoya and Tomita, Koichi and Kobayashi, Yuki and Kuroda, Kazuya and Koyama, Yoshihisa and Kobayashi, Hikaru and Kubo, Tateki
Journal: Scientific reports (2022): 1--8
Pre-treatment with curcumin ameliorates cisplatin-induced kidney damage by suppressing kidney inflammation and apoptosis in rats
Authors: Soetikno, Vivian and Sari, Shinta Dewi Permata and Maknun, Lulu Ul and Sumbung, Nielda Kezia and Rahmi, Deliana Nur Ihsani and Pandhita, Bashar Adi Wahyu and Louisa, Melva and Estuningtyas, Ari
Journal: Drug research (2019): 75--82
Authors: Soetikno, Vivian and Sari, Shinta Dewi Permata and Maknun, Lulu Ul and Sumbung, Nielda Kezia and Rahmi, Deliana Nur Ihsani and Pandhita, Bashar Adi Wahyu and Louisa, Melva and Estuningtyas, Ari
Journal: Drug research (2019): 75--82
References
View all 21 references: Citation Explorer
Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: Analytical and biological challenges.
Authors: Tsikas D., undefined
Journal: Anal Biochem (2016)
Authors: Tsikas D., undefined
Journal: Anal Biochem (2016)
Formation of Malondialdehyde, 4-Hydroxynonenal, and 4-Hydroxyhexenal during in Vitro Digestion of Cooked Beef, Pork, Chicken, and Salmon
Authors: Steppeler C, Haugen JE, Rodbotten R, Kirkhus B.
Journal: J Agric Food Chem (2016): 487
Authors: Steppeler C, Haugen JE, Rodbotten R, Kirkhus B.
Journal: J Agric Food Chem (2016): 487
Development, validation and biomedical applications of stable-isotope dilution GC-MS and GC-MS/MS techniques for circulating malondialdehyde (MDA) after pentafluorobenzyl bromide derivatization: MDA as a biomarker of oxidative stress and its relation to 15
Authors: Tsikas D, Rothmann S, Schneider JY, Suchy MT, Trettin A, Modun D, Stuke N, Maassen N, Frolich JC.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2016): 95
Authors: Tsikas D, Rothmann S, Schneider JY, Suchy MT, Trettin A, Modun D, Stuke N, Maassen N, Frolich JC.
Journal: J Chromatogr B Analyt Technol Biomed Life Sci (2016): 95
Plasma malondialdehyde as biomarker of lipid peroxidation: effects of acute exercise
Authors: Spirl, undefined and eli AL, Deminice R, Jordao AA.
Journal: Int J Sports Med (2014): 14
Authors: Spirl, undefined and eli AL, Deminice R, Jordao AA.
Journal: Int J Sports Med (2014): 14
A specific, accurate, and sensitive measure of total plasma malondialdehyde by HPLC
Authors: Moselhy HF, Reid RG, Yousef S, Boyle SP.
Journal: J Lipid Res (2013): 852
Authors: Moselhy HF, Reid RG, Yousef S, Boyle SP.
Journal: J Lipid Res (2013): 852
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