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

Cat. No.: M1508248

GRADE & PURITY BioReagent

Overview

When animals 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. Some other biochemical reactions in organisms also produce MDA, such as catalysis by thromboxane synthase. However, as long as appropriate controls are set during the assay, changes in lipid oxidation levels can be observed.

Detection Principle

Under conditions of high temperature and in an acidic environment, MDA can react with TBA to form a red MDA-TBA adduct. The MDA-TBA adduct has a maximum absorption at 535 nm, allowing detection via colorimetry. Additionally, the MDA-TBA adduct can also be excited at 535 nm, producing a maximum emission wavelength at 553 nm, enabling fluorescence detection.

Applicable Samples: Plasma, serum, urine, animal/plant tissues, or cell lysates.

Reagents, consumables and Equipments not provided

• Spectrophotometer or microplate reader (capable of measuring absorbance at 535 nm)

• Centrifuge tubes or 96-well plate, Centrifuge, Water bath or incubator

• Physiological saline or PB

Procedure

1. Sample Preparation

1.1 Serum, Plasma, Urine, Cerebrospinal Fluid Samples

Serum or plasma separated from the test sample should be free of hemolysis. Assay directly. If exceeding the linear range, dilute with physiological saline or PBS before assay.

1.2 Tissue, Cell, and Other Samples

Tissues or cells can be homogenized or lysed using PBS or RIPA lysis buffer, etc. 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 4°C, 8,000~12,000 g for 10 minutes. Collect the supernatant for subsequent assays. Steps such as homogenization or lysis should be performed on ice or at 4°C.

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):

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 the TBA Diluent. For example, take 68 mg TBA and dilute with 10 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. The prepared TBA working solution should be stored at 4°C protected from light and is valid for at least one month.

3. Dilution of Series Standards

Take an appropriate amount of MDA Standard (1 mmol/L) and dilute it with a suitable solution to concentrations of 1, 2, 5, 10, and 20 µM (if performing a simple rapid assay, dilute the standard directly to 10 µM).

Note: When the test sample is serum or plasma, the standard should preferably be diluted with physiological saline. When the test sample is obtained from homogenate, lysate, or PBS, the standard should preferably be diluted with the same solution. The principle is to ensure comparability among the blank, standard, and test tubes. Prepared MDA standards can be stored at 4°C protected from light and are valid for at least 3 months.

4. Preparation of MDA Assay Working Solution

Immediately before detection, prepare a sufficient amount of fresh MDA Assay Working Solution according to the number of samples to be tested (including controls), as a reference:

Number of Assays	1 time	10 times	20 times
TBA Working Solution	0.75ml	7.5ml	15ml
Antioxidant	0.031ml	0.31ml	0.62ml
MDA Assay Solution	0.075ml	0.75ml	1.5ml
MDA Separation Solution	2.25ml	22.5ml	45ml
Total Volume	3.106ml	31.06ml	62.12ml

5. MDA Sample Loading

Add 0.08 mL of an appropriate solution to a centrifuge tube or other suitable container as a blank control (Note: When the test sample is serum or plasma, the standard should preferably be diluted with physiological saline. When the test sample is obtained from homogenate, lysate, or PBS, the standard should preferably be diluted with the same solution. The principle is to ensure comparability among the blank, standard, and test tubes.). Add 0.08 mL of the above series of different concentration standards to generate a standard curve (if performing a simple rapid assay, directly add the 10 µM standard). Add 0.08 mL of sample for measurement. Then add 3 mL of MDA Assay Working Solution. The assay reaction system can be set up as follows, adding reagents sequentially:

Additive (mL)	Blank Tube	Standard Tube	Test Tube
Homogenate, lysate, PBS, physiological saline, etc.	0.08	－	－
Standard	－	0.08	－
Test Sample	－	－	0.08
MDA Assay Working Solution	3	3	3

Mix well, cap, and boil in a 95°C water bath for 40 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.

6. MDA Assay

Cool in water or under running water to room temperature. Centrifuge at 3,000 rpm for 15 minutes or 4,000 rpm for 10 minutes. Take the supernatant, which will be yellow to reddish-brown in color. Zero the instrument with distilled water. Using a cuvette with a 1 cm light path, measure the absorbance of the blank, standard, and test tubes at 535 nm with a spectrophotometer. If not convenient, absorbance between 530~540 nm can also be measured. Record the values as A_Blank, A_Standard, A_Test.

7. Calculation of Results

If performing a simple rapid assay, calculate directly using the 10 µM standard to obtain the molar concentration of MDA. If precise calculation is required, use the MDA standard concentrations as the x-axis and the corresponding absorbance values as the y-axis to plot a standard curve. Calculate the concentration of MDA in the extract (C1, µmol/L) based on the standard curve. For solid tissues, the initial MDA content in the sample (C2, µmol/g) can be expressed per unit weight of protein or tissue weight, e.g., µmol/g protein or tissue.

Formula for Simple Rapid Calculation of MDA Content in Liquid Samples (Serum, Plasma, Urine, etc.):

MDA Concentration (µmol/L) = C1 = (A_Test - A_Blank) / (A_Standard - A_Blank) × 10

Formula for Simple Rapid Calculation of MDA Content in Cell or Tissue Samples:

MDA Content (µmol/g) = C2 = (A_Test - A_Blank) / (A_Standard - A_Blank) × 10 / Protein Concentration (mg/mL)= C1 / Protein Concentration (mg/mL)

or

MDA Content (µmol/g) = C2 = (A_Test - A_Blank) / (A_Standard - A_Blank) × 10 / [w / (V_Total × 10⁻³)]= C1 / [w / (V_Total × 10⁻³)]

Parameter Description:

A_Test: Absorbance of the test tube

A_Standard: Absorbance of the standard tube

A_Blank: Absorbance of the blank tube

w: Mass of tissue sample (g)

V_Total: Total volume of sample extraction solution (mL)

Reference Standard Curve Range

When measuring the MDA standard at 10 µM, its absorbance measured by a microplate reader is mostly between 1.3~2.3 (without zeroing). Measure the absorbance of MDA standards at 1, 2, 4, 6, 8, 10, 12, and 14 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 16 µmol/L.

Notes

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

2. Recommended sample amounts: Serum, plasma, urine: take 0.1 mL; Low-density lipoprotein suspension: take 0.1~0.2 mL; Edible oil: take 0.03 mL; Liver, myocardium, muscle, etc.: take 0.1~0.2 mL of 5% or 10% homogenate.

3. If the measured sample absorbance is low, the water bath time can be extended to 80 minutes, but this should be done consistently for all samples to avoid batch-to-batch variation.

4. If test samples cannot be assayed promptly, store at -20°C. They are stable for 4 days.

5. Avoid using anticoagulants such as EDTA, citrate, sodium fluoride, oxalate, etc.

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

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

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. M1508248A: Store at room temperature long term (12 months). Store in the dark. M1508248B, M1508248E, M1508248F: Store at room temperature long term (12 months). M1508248C, M

Contents & Storage

M1508248	Component	50T	100T	Storage
M1508248A	TBA	0.4g	0.8g	RT. Store in the dark.
M1508248B	TBA Diluent	50 mL	100 mL	RT.
M1508248C	Antioxidant	2.5 mL	5 mL	-20℃. Store in the dark.
M1508248D	MDA Standard (1 mmol/L)	0.2 mL	0.4 mL	-20℃. Store in the dark.
M1508248E	MDA Assay Solution	5 mL	10 mL	RT.
M1508248F	MDA Separation Solution	75 mL×2	150 mL×2	RT.