Plant Malondialdehyde (MDA) Content Assay Kit (TBA, Colorimetric Method)

Cat. No.: P1508323

GRADE & PURITY BioReagent

Overview

When animal or plant cells undergo oxidative stress, lipid oxidation occurs. Malondialdehyde (MDA) is a natural product of lipid oxidation in organisms. After some fatty acids oxidize, they 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. This kit is designed for the detection of MDA in plant tissues (roots, stems, leaves, seeds, etc.) and is specifically used for assessing plant lipid peroxidation levels. It is not suitable for animal tissues, cells, blood, etc.

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 a maximum absorption at 532 nm, with a molar extinction coefficient of 155 mmol/(L·cm), and a minimum absorption at 600 nm. Sugars in plant tissues can interfere with the MDA-TBA reaction. We have developed an empirical formula to eliminate this interference. Alternatively, the content can be determined by comparison with a standard.

Applicable Samples: Plant tissues

Reagents, consumables and Equipments not provided

• Spectrophotometer or microplate reader (capable of measuring absorbance at 532 nm)

• Water bath or incubator, Centrifuge

• Scissors, Centrifuge tubes, Small test tubes or 96-well plate

• Plant roots, stems, leaves, etc.

Procedure

1. Sample Preparation

Preparation of MDA Extract: Take an appropriate amount of plant roots, stems, leaves, seeds, etc., weigh, cut into small pieces, and add Tissue Homogenization Buffer at a ratio of 4 mL per 0.4 g of plant sample. Homogenize thoroughly (generally, 0.4–1 g of plant sample is sufficient). Centrifuge at 4,000 g for 10 minutes. Collect the supernatant for use; this supernatant is the MDA extract. If using a microplate reader for detection, reduce the scale of extract preparation accordingly, e.g., take 0.2 g of plant sample and add 2 mL of Tissue Homogenization Buffer.

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. Measuring protein concentration is not a mandatory step; the empirical formula can also be used for calculation.

Compatibility of Common Chemical Components in Samples with this Kit (Reference Table)

Reagent Category	Chemical Component	Interference?
Buffer	HEPES (100mM)	No
Buffer	Borate (50mM)	No
Buffer	Phosphate (100mM)	No
Buffer	Tris (25mM)	No
Detergent	CHAPS (≤1%)	No
Detergent	Triton X-100 (≤1%)	No
Detergent	Tween 20 (≤1%)	No
Inhibitor/Chelator	PMSF (≤200μM)	No
Inhibitor/Chelator	EDTA (≤1mM)	No
Inhibitor/Chelator	EGTA (≤1mM)	No
Inhibitor/Chelator	Antipain (≤100μg/ml)	No
Inhibitor/Chelator	Chymostatin (≤10μg/ml)	No
Inhibitor/Chelator	Leupeptin (≤10μg/ml)	No
Inhibitor/Chelator	Trypsin (≤10μg/ml)	No
Other	Glycerol (≤10%)	No
Other	Sucrose (250mM)	Yes

2. Preparation of TBA Working Solution

Weigh an appropriate amount of TBA and prepare a 0.68% TBA working solution using Tissue Homogenization Buffer. For example, take 0.068 g TBA and dilute with 10 mL Tissue Homogenization Buffer 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. The prepared TBA working solution should be stored at 4°C protected from light and is valid for at least one month.

3. Standard Dilution

• For simple rapid assay: Dilute the standard directly to 10 µM.

• For precise assay: Dilute an appropriate amount of standard with Tissue Homogenization Buffer to 1, 2, 5, 10, 20, and 50 µM.

• For calculation using the empirical formula: No standard is needed.

• Prepared MDA standards can be stored at 4°C protected from light and are valid for at least 3 months.

4. Sample Assay

4.1 Operation Table 

(1) Spectrophotometer Assay

Add 1 mL of Tissue Homogenization Buffer to a centrifuge tube or other suitable container as a blank control. Add 1 mL of MDA extract for measurement. Then add 1 mL of TBA working solution. The assay reaction system can be set up as follows, adding reagents sequentially:

Additive (mL)	Blank Tube	Standard Tube	Test Tube
Tissue Homogenization Buffer	1	－	－
MDA Extract	－	－	1
Antioxidant	0.005	0.005	0.005
TBA Working Solution	1	1	1

(2) Microplate Reader Assay

Add 200 µL of Tissue Homogenization Buffer to a centrifuge tube or other suitable container as a blank control. Add 200 µL of MDA extract for measurement. Then add 200 µL of TBA working solution. The assay reaction system can be set up as above, adding reagents sequentially (add 5 µL of Antioxidant).

Additive (µL)	Blank Tube	Standard Tube	Test Tube
Tissue Homogenization Buffer	200	－	－
MDA Extract	－	－	200
Antioxidant	5	5	5
TBA Working Solution	200	200	200

4.2 Mix well, cover, and boil in a 95°C water bath for 30 minutes. 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 with a heated lid or a 0.5 mL PCR instrument.

4.3 Cool in a cold water bath or under running water to room temperature. Centrifuge at 4,000 g for 10 minutes.

4.4 Take the supernatant. Zero the instrument with distilled water. Measure the absorbance at 532 nm using a spectrophotometer or microplate reader. If not convenient, absorbance between 530~540 nm can also be measured. Record the values as A_Blank, A_Standard, A_Test. If using the empirical formula for calculation, measure the absorbance at 450 nm, 532 nm, and 600 nm, recorded as A₄₅₀, A₅₃₂, A₆₀₀.

5. Calculation of Results

• For simple rapid assay: Calculate directly using the 10 µM standard to obtain the molar concentration of MDA.

• When using the empirical formula: No standard curve or standard measurement is needed.

• For precise calculation: Plot a standard curve with MDA standard concentrations as the x-axis and the corresponding absorbance values as the y-axis. Calculate the concentration of MDA in the extract based on the standard curve.

• For solid tissues, the initial MDA content in the sample can be expressed per unit weight of protein or tissue weight, e.g., µmol/g protein or µmol/g tissue.

5.1 Simple Rapid MDA Content Calculation Formula:

MDA Content (µmol/g protein) = (A_Test - A_Blank) / (A_Standard - A_Blank) × C_Standard / Cpr

Parameter Description:

A_Test: Absorbance of the test sample at 532 nm

A_Standard: Absorbance of the standard at 532 nm

A_Blank: Absorbance of the blank control at 532 nm

C_Standard: Standard concentration, 10 µmol/L

Cpr: Protein mass concentration, mg/mL = g/L

5.2 Empirical Formula (without using a standard):

MDA Concentration (µmol/L) = 6.45 × (A₅₃₂ - A₆₀₀) - 0.56 × A₄₅₀

MDA Content (µmol/g tissue) = MDA Concentration (µmol/L) × Volume of MDA Extract (L) / Fresh Weight of Plant Tissue (g)

Parameter Description:

A₅₃₂: Absorbance of the test sample at 532 nm

A₆₀₀: Absorbance of the test sample at 600 nm

A₄₅₀: Absorbance of the test sample at 450 nm

Appendix

Reference Standard Curve Range: When measuring the MDA standard at 10 µM, its absorbance measured by a spectrophotometer is mostly between 1.4~1.8. Measure the absorbance of MDA standards at 1, 2, 5, 10, and 20 µM to generate a standard curve.

Note: Due to differences in detection instruments and operational techniques, the reference range may vary. This value is for reference only. For accurate calculation of MDA content, multi-point determination can be performed. Based on assay experience, the standard curve may deviate for standard concentrations below 2 µmol/L and above 50 µmol/L.

Notes

1. Avoid repeated freeze-thaw cycles for the aforementioned low-temperature reagents to prevent loss of efficacy or reduced efficiency.

2. If a spectrophotometer is not available, a microplate reader can also be used, but the required sample volume will increase accordingly.

3. Test samples should be as fresh as possible. After extraction, assay should be performed promptly to avoid decreased activity.

4. If the MDA extract cannot be assayed promptly, store at -20°C. It is stable for 4 days.

5. For your safety and health, please wear a lab coat and disposable gloves during operation.

6. Use reagents as soon as possible after opening to avoid affecting subsequent experimental results.

7. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.

Storage and Shipping

Storage

Protected from light,Room temperature,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions. P1508323A, P1508323B: Store at room temperature long term (12 months). Store in the dark. P1508323C, P1508323D: Store at -20℃ long term (12 months). Store in the dark.

Contents & Storage

P1508323	Component	50T	100T	Storage
P1508323A	Tissue Homogenization Buffer	250 mL	500 mL	RT. Store in the dark.
P1508323B	TBA	0.35 g	0.7 g	RT. Store in the dark.
P1508323C	Antioxidant	0.5 mL	1 mL	-20℃. Store in the dark.
P1508323D	MDA Standard (1 mmol/L)	0.5 mL	1 mL	-20℃. Store in the dark.