This kit can be used to measure the MDA content in serum, plasma, tissue and cell samples.

Detection significance

The body produce oxygen free radicals through the enzyme system and non-enzyme system, which can attack unsaturated fatty acid on biofilm and lead to lipid peroxidation and form lipid peroxide, such as aldehyde group (MDA), keto-, hydroxyl, carbonyl, etc. Oxygen free radicals cause cell damage not only by peroxidation of polyunsaturated fatty acids in biofilm, but also by decomposition products of lipid hydroperoxide. Detection of the MDA content can reflect the level of lipid peroxidation in cells and reflect level of cellular damage indirectly.

Detection principle

MDA in the catabolite of lipid peroxide can react with thiobarbituric acid (TBA) and produce red compound, which has a maximum absorption peak at 532 nm.

Experiment instruments

Test tube

Micropipettor

Vortex mixer

Water bath

Spectrophotometer (532 nm)

Sample preparation

1.  Serum/plasma or Cell supernatant: Detect the sample directly. If the concentration is beyond the linear range, then dilute the sample with saline before detection.

2.  Tissue: Mince the tissues to small pieces, then weigh and homogenize in PBS(0.01 M, pH 7.4) on ice, the volume of PBS (mL): the weight of the tissue (g) =9:1. The tissue homogenate is centrifuged at 10000 g for 10 min. Collect the supernatant and preserve it on ice for detection. Meanwhile, determine the concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

3.  Cells：Collect 1×106 cells and add 300-500 μL of cold PBS (0.01M, pH7.4), then treat the sample with mechanical homogenate or sonication on ice. The cell homogenate is centrifuged at 10000 g for 10 min. Collect the supernatant and preserve it on ice for detection. Meanwhile, determine the concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).

Operation steps

1.  Standard operation table

Note: 1. a * represents the volume of sample, standard, absolute ethanol and reagent 1, they are equal.

For example, the sampling volume is 0.1 mL, the volume of standard, absolute ethanol and reagent 1 are 0.1 mL for each. If the sampling volume is 0.2 mL, the volume of standard, absolute ethanol and reagent 1 are 0.2 mL for each.

  2. In general, 1~2 tubes of standard and blank are established for each batch. If the serum (plasma) samples are no hemolysis or lipidemia, control tube can be remove, just need to establish blank tube.

  3. Reference sampling volume:

Serum (plasma): 0.1~0.2 mL,  

      Low density lipoprotein suspension: 0.1~0.2 mL,

      Liver tissue, myocardium, muscle tissue: 5% or 10% tissue homogenate, 0.1~0.2 mL.

Technical parameter

1. The sensitivity of the kit is 0.38 nmol/mL.

2. The intra-assay CV is 4.9% and the inter-assay CV is 8%.

3. The recovery of the kit is 101%.

4. The detection range of the kit is 0.38-133.33 nmol/mL.

Notes

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The valid of kit is 6 months.

4. Do not use components from different batches of kit.

5. It is recommended to use disposable EP tubes or clean glass tubes with stopper for heating.

6. The temperature of water-bath and the time of incubation should be stabilized.

7. The supernatant for assay should not contain sediment, otherwise it will affect the OD values. It is recommended to use a pipette to take the supernatant.

Citations