Malondialdehyde (MDA) Content Assay Kit/SLBC0025
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Product Detail
Malondialdehyde (MDA) Content Assay Kit
Note: It is necessary to predict 2-3 large difference samples before the formal determination.
Operation Equipment: Spectrophotometer/Microplate reader
Cat No: SLBC0025
Size: 100T/96S
Components:
Extraction reagent: Liquid 110 mL×1. Storage at 4℃.
Reagent I: Liquid 42 mL×1. Storage at 4℃.
Reagent Ⅱ: Powder×2. Storage at 4℃.
MDA working reagent: add 20 mL Reagent I to Reagent Ⅱ, dissolve (heat at 70℃ or with ultrasonic) and mix thoroughly. Storage at 4℃.
Reagent Ⅲ: Liquid 12 mL×1. Storage at 4℃.
Note: The working solution for MDA detection is difficult to dissolve, which can be heated at 70℃ and vibrated violently to promote dissolution. Or by ultrasonic treatment to promote dissolution.
Product Description:
Lipid peroxide is produced by the action of oxygen free radicals on unsaturated fatty acid, then resolves to compounds, including malondialdehyde (MDA). The level of lipid peroxidation can be showed by detecting the level of MDA.
Under acidic and high temperature conditions, the brown red 3,5,5- three methyl sulfamethoxazole -2,4-two ketone is synthesized with MDA and thiobarbituric acid (TBA) taking place condensation reaction, and the largest absorption wavelength is 532 nm. After colorimetry, the MDA content in the sample can be estimated.
Reagents and Equipment Required but Not Provided:
Spectrophotometer/microplate reader, water bath, centrifuge, transferpettor, micro glass cuvette/96 well flat-bottom plate, mortar/homogenizer, ice and distilled water.
Procedure:
I. Sample preparation
1. Bacteria or cells:
Collect bacteria or cells into the centrifuge tube. 5 million bacteria or cells could be mixed with 1 mL of Extraction reagent. Use ultrasonication to split bacteria and cells (placed on ice, ultrasonic power 200W, ultrasonic time 3 seconds, interval 10 seconds, repeat for 30 times). Centrifuge at 8000 ×g for 10 minutes at 4℃ to remove insoluble materials, and take the supernatant on ice before testing.
2. Tissue sample:
0.1 g of tissue could be mixed with 1 mL of Extraction reagent and fully homogenized on ice bath. Then centrifuge at 8000 ×g for 10 minutes at 4℃ to remove insoluble materials, and take the supernatant on ice before testing.
3. Serum: Detect directly.
II. Determination procedure:
1. Preheat the spectrophotometer for 30 minutes, set zero with distilled water.
2. Add reagents with the following list:
Reagent (μL) | Test tube (T) | Blank tube(B) |
MDA working reagent | 300 | 300 |
Sample | 100 | - |
Distilled water | - | 100 |
Reagent Ⅲ | 100 | 100 |
The mixture would be incubated at 100℃ for 60 minutes (tightly close to prevent moisture loss), cooled on ice, and centrifuged at 10000 ×g for 10 minutes at room temperature to remove insoluble materials. Take 200μL of supernatant in micro glass cuvette/96 well flat-bottom plate, and measure the absorbance at 532 nm and 600 nm. ∆A532=A532(T)-A532(B), ∆A600 =A600(T)-A600(B), ΔA=ΔA532-ΔA600.
Blank tube needs to test once or twice.
III. Calculation:
A 96 well plate
1. Tissue, bacteria or cultured cells
1) Protein concentration:
MDA (nmol/ mg prot) =[ΔA×Vrv÷（ε×d）×109]÷(Cpr×Vs)= 53.763×ΔA÷Cpr
2) Sample weight:
MDA (nmol/g weight)=[ΔA×Vrv÷（ε×d）×109]÷(W×Vs÷Vsv)= 53.763×ΔA÷W
3) Cell amount:
MDA (nmol/104cell)= [ΔA×Vrv÷（ε×d）×109] ÷(500×Vs÷Vsv)=0.1075×ΔA
4) Serum (plasma) sample
MDA含量（nmol/mL）=[ΔA×Vrv÷（ε×d）×109]÷Vs=53.763×ΔA
Vrv: Total reaction volume, 5×10-4L ;
ε: Molar extinction coefficient, 1.55×105 L/mol/cm
d: light path of 96-well plate, 15px
Vs: Sample volume, 0.1 mL;
Vsv: The volume of Extraction reagent, 1 mL;
Cpr: Sample protein concentration, mg/mL;
W: Sample weight, g;
500: Total number of bacteria and cells, 5 million.
B． Micro glass cuvette
1) Protein concentration:
MDA (nmol/mg prot)= [ΔA×Vrv÷（ε×d）×109]÷(Cpr×Vs)=32.258×ΔA÷Cpr
2) Sample weight:
MDA (nmol/g weight)= [ΔA×Vrv÷（ε×d）×109]÷(W×Vs÷Vsv)= 32.258×ΔA÷W
3) Cell amount:
MDA (nmol/104cell)= [ΔA×Vrv÷（ε×d）×109] ÷(500×Vs÷Vsv)=0.0645×ΔA
4) Serum:
MDA (nmol/mL)= [ΔA×Vrv÷（ε×d）×109]÷Vs=32.258×ΔA
Vrv: Total reaction volume, 5×10-4L ;
ε: Molar extinction coefficient, 1.55×105 L/mol/cm
d: light path of 96-well plate, 15px
Vs: Sample volume, 0.1 mL;
Vsv: The volume of Extraction reagent, 1 mL;
Cpr: Sample protein concentration, mg/mL;
W: Sample weight, g;
500: Total number of bacteria and cells, 5 million.
Note:
If it is found that the absorbance value of the sample is too low, the boiling water bath time can be adjusted from 60 minutes to 90 minutes or longer. The detection of MDA in the same experiment needs to be extended to the same time to avoid errors.
Experimental example:
1.The plasma is taken and operated according to the determination steps. The absorbances are measured by 96 well plate. ΔA450 = A450T- A450B= 0.320-0.052 = 0.268, ΔA532 = A532T- A532B= 0.168-0.046 = 0.122, ΔA600 =A600T- A600B= 0.093-0.043 = 0.05
MDA content (nmol/mL) = 5 × (12.9 × (ΔA532 - ΔA600) - 2.58 × ΔA450) = 1.1868 nmol/mL.
2. 5 million HeLa cells are collected and treated with 1 mL of Extraction reagent. After centrifugation, the supernatant is determined according to the determination steps. The absorbances are measured by 96 well plate. ΔA450 = A450T- A450B= 0.097-0.052 = 0.045, ΔA532 = A532T- A532B= 0.101-0.046 = 0.055, ΔA600 = A600T-A600B= 0.043-0.043 = 0.0
MDA content (nmol/104 cell) = 0.01×(12.9×(ΔA532 - ΔA600) - 2.58 × ΔA450) = 0.01 × (12.9 × (0.055-0) - 2.58 × 0.045) = 0.0059 nmol/104 cell
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