This kit can be used to measure MDA content in serum, plasma, tissue, culture cells, bacteria and other samples.
The body produce oxygen free radicals through the enzyme system and non-enzyme system, which can attack unsaturated fatty acid on biofilm, leading to lipid peroxidation and forming lipid peroxide, such as aldehyde group (MDA), keto-, hydroxyl, carbonyl, etc. Lipid peroxide can not only transform active oxygen to active chemical, but also enhance the effect of active oxygen. Detection of the MDA content can reflect the level of lipid peroxidation in cells and reflect level of cellular damage indirectly.
MDA in the catabolite of lipid peroxide can react with thiobarbituric acid (TBA) and present red compound, which has a maximum absorption peak at 532 nm, thus the lipid peroxide (LPO) content can be calculated after the colorimetric determination. Then measure the absorbance at 600 nm, and the MDA content can be calculated according to the difference of absorbance value at 532 nm and 600 nm.
Micro quartz cuvette/Microplate (96 wells), Mortar, Micropipettor, Vortex mixer, Water-bath, Centrifuge, Spectrophotometer/Microplate Reader
Extraction of MDA
1. Preparation of bacteria or cells samples:
Bacteria or cells: Collect the bacteria or cultured cells samples into a centrifuge tube, then centrifuge the sample and discard the supernatant. Add Extract Solution into the sediment according to the ratio of Bacteria or Cells number (104): Extract Solution (mL) =500~1000: 1(it is recommended to add 1 mL of Extract Solution into 5×106 bacteria or cells), then treat the sample with sonication on ice (power: 20% or 200W, 3 seconds/time, interval for 10 seconds, repeat for 30 times). Centrifuge at 8000 g for 10 min at 4℃. Take the supernatant and preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165)
2. Preparation of tissue samples:
Add Extract Solution into the tissue sample according to the ratio of Weight (g): Extract Solution (mL) =1: 5~10 (it is recommended to add 1 mL of Extract Solution into 0.1 g of tissue), then homogenize the sample on ice. Centrifuge the tissue homogenate at 8000 g for 10 min at 4℃. Take the supernatant and preserve it on ice for detection. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165)
3. Serum / plasma samples:
Detect the serum or plasma samples directly.
1. Add 0.3 mL of Reagent 2 into a 1.5 mL centrifuge tube, then add 0.1 mL of sample, mix fully.
2. Incubate at 95℃ for 30 min (cover the cap to prevent the loss of water), then cool the tubes in ice-bath. Centrifuge at 10000 g for 10 min at 25℃.
3. Take 200 μL of the supernatant into the micro quartz cuvette or 96 wells Microplate. Measure the absorbance value at 532 nm and 600 nm. △A= A532 - A600.
1. The kit is for scientific research only.
2. Instructions should be followed strictly, changes of operation may result in unreliable results.
3. The validity of kit is 3 months.
4. Do not use components from different batches of kit.
5. If the MDA content is calculated by concentration, the protein concentration of the sample needs to be determined separately (E-BC-K318, E-BC-K168, E-BC-K165).