BioReagent BioReagent for sensitive chromatographic and analytical workflows requiring minimal baseline interference.
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Cited in 0 peer-reviewed publications across chromatography, organic synthesis, and cross-coupling reactions.
When animal or plant cells undergo oxidative stress, lipid oxidation occurs. Malondialdehyde (MDA) is a natural product of lipid oxidation in organisms. Some fatty acids oxidize and gradually decompose into a series of complex compounds including MDA. At this point, the level of lipid oxidation can be detected by measuring MDA levels. Therefore, the determination of MDA is widely used as an indicator of lipid oxidation. Other biochemical reactions in organisms also produce MDA, such as catalysis by thromboxane synthase. However, by setting appropriate controls during the assay, changes in lipid oxidation levels can be observed.
Detection Principle
Under conditions of high temperature and in an acidic environment, MDA reacts with TBA to form a red MDA-TBA adduct. The MDA-TBA adduct has maximum absorption at 553 nm, with an excitation wavelength of 515 nm, allowing detection via fluorescence colorimetry.
Applicable Samples: Plasma, serum, urine, animal/plant tissues, or cell lysates.
| M1508267 | Component | 100T | Storage |
| M1508267A | MDA Precipitation Solution | 50 mL | RT. |
| M1508267B | Phosphotungstic Acid Solution | 50 mL | RT. Store in the dark. |
| M1508267C | MDA Standard (1 mmol/L) | 0.4 mL | -20℃. Store in the dark. |
| M1508267D | TBA | 0.8 g | RT. Store in the dark. |
| M1508267E | TBA Diluent | 100 mL | RT. |
| M1508267F | Antioxidant | 5 mL | -20℃. Store in the dark. |
| M1508267G | MDA Separation Solution | 100 mL×4 | RT. Store in the dark. |
Reagents, consumables and Equipments not provided
Fluorescence spectrophotometer or fluorescence microplate reader
Centrifuge, water bath or incubator
Centrifuge tubes, small test tubes, or 96-well plate
Distilled water
Procedure
1. Sample Preparation
1.1 Serum, Plasma, Urine, Cerebrospinal Fluid Samples
The serum or plasma separated from the test sample should be free of hemolysis. Take 20 µl of the liquid test sample, sequentially add 0.5 mL MDA Precipitation Solution, 3.5 mL distilled water, and 0.5 mL Phosphotungstic Acid Solution. Mix well, let stand at room temperature for 5 minutes, centrifuge at 3,500 rpm for 10 minutes, and discard the supernatant. Add 1 mL distilled water to the precipitate, vortex for 2 minutes to fully dissolve the precipitate (MDA sample) to obtain the MDA test solution.
1.2 Tissue, Cell, and Other Samples
Tissues or cells can be homogenized or lysed using PBS or Western & IP cell lysis buffer, etc. When homogenizing or lysing tissue, the tissue weight should constitute 10% of the homogenization or lysis buffer. For cells, use 0.1 mL of lysis buffer or homogenization buffer per 10⁶ cells. After homogenization or lysis, centrifuge at 1,600 rpm for 10 minutes and take the supernatant for subsequent assays. Steps such as homogenization or lysis should be performed on ice or at 4°C. Take 20 µl of the supernatant extracted after homogenization, sequentially add 0.5 mL MDA Precipitation Solution, 3.5 mL distilled water, and 0.5 mL Phosphotungstic Acid Solution. Mix well, let stand at room temperature for 5 minutes, centrifuge at 3,500 rpm for 10 minutes, and discard the supernatant. Add 1 mL distilled water to the precipitate, vortex for 2 minutes to fully dissolve the precipitate (MDA sample) to obtain the MDA test solution.
*Note: After sample preparation, the protein concentration can be measured using a BCA Protein Assay Kit (B665595/R1491648) to facilitate subsequent calculation of MDA content per unit protein weight in tissues or cells.*
1.3 Compatibility of Common Chemical Components in Samples with this Kit (Reference Table)
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2. Standard Dilution
Take an appropriate amount of MDA Standard (1 mmol/L) and dilute with distilled water to 0.5, 1, 2, 5, and 10 µM (if performing a simple rapid assay, dilute the standard directly to 0.5 µM).
3. Preparation of TBA Working Solution
Weigh an appropriate amount of TBA and prepare a 0.68% TBA working solution using the TBA Diluent. For example, take 34 mg TBA and dilute with 5 mL TBA Diluent to obtain a final concentration of 0.68% TBA working solution. The TBA working solution must be completely dissolved before use. Heating to 60°C can aid dissolution, and repeated vigorous vortexing can also help.
4. MDA Sample Loading
Set up the assay reaction system in centrifuge tubes or other suitable containers according to the table below, adding reagents sequentially:
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Mix well, cover, and boil accurately in a 95°C water bath for 60 minutes (do not disturb). Take care to avoid violent boiling and splashing during heating. If using a heat block for heating, ensure the tube caps are tightly pressed with a weight. If using a boiling water bath, use centrifuge tubes with lockable caps or screw-cap tubes, or seal the tube mouth with Parafilm and pierce a small hole with a needle. The most convenient and accurate heating method is to use a metal bath instrument with a heated lid.
5. MDA Assay
Cool in water or under running water to room temperature. Add 3.5 mL MDA Separation Solution, shake and extract for 1 minute, centrifuge at 3,000 rpm for 5 minutes, and take the supernatant. Zero the instrument with distilled water. Measure the fluorescence intensity using a fluorescence spectrophotometer or fluorescence microplate reader with excitation at 515 nm and emission at 553 nm.
6. Calculation of Results
For samples such as plasma, serum, or urine, plot a standard curve with MDA standard concentration as the x-axis and the corresponding fluorescence intensity as the y-axis. Calculate the concentration of MDA in the extract based on the standard curve. If performing a simple rapid assay, calculate directly by multiplying by the 0.5 µM standard to obtain the molar concentration of MDA. For cell or tissue samples, after calculating the MDA content in the sample solution, the initial MDA content in the sample can be expressed per unit weight of protein or tissue weight, e.g., µmol/mg protein or µmol/mg tissue.
Formula for Simple Rapid Calculation of MDA Content in Liquid Samples (Serum, Plasma, Urine, etc.):
MDA Concentration (µmol/L) = (ATest - ABlank) / (AStandard - ABlank) × 25
Formula for Simple Rapid Calculation of MDA Content in Cell or Tissue Samples:
MDA Concentration (µmol/mg) = (ATest - ABlank) / (AStandard - ABlank) × 25 / Protein Concentration (mg/mL)
Parameter Description:
ATest: Fluorescence intensity of the test well
A<sub>Standard</sub>: Fluorescence intensity of the standard well
ABlank: Fluorescence intensity of the blank well
Notes
Avoid repeated freeze-thaw cycles for the aforementioned low-temperature reagents to prevent loss of efficacy or reduced efficiency.
Recommended sample amounts: Serum, plasma, urine: take 20 µL; Low-density lipoprotein suspension: take 20–40 µL; Edible oil: take 30 µL; Liver, myocardium, muscle, etc.: take 20–40 µL of 5% or 10% homogenate.
If test samples cannot be assayed promptly, store at -20°C. They are stable for 4 days.
Avoid using anticoagulants such as EDTA, citrate, sodium fluoride, oxalate, etc.
Diluted MDA standards can be stored at 4°C protected from light and are valid for 3 months.
TBA working solution should be stored at 4°C protected from light and is valid for 1 month.
During the MDA assay step, if turbidity appears when extracting the upper layer after centrifugation, add 1 drop of anhydrous ethanol.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Use reagents as soon as possible after opening to avoid affecting subsequent experimental results.
This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
Comprehensive hazard, handling, storage, and regulatory compliance document.
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E2606001