Application
This kit can be used to detect animal serum, plasma, tissue and leucocyte, etc.
Detection principle
The kit can simulate xanthine and xanthine oxidase reaction system to generate superoxide anion free radical O2-, then add Electron transfer material and color developing agent, the reaction system turns aubergine, measure the OD value with a spectrophotometer. When the superoxide anion radical inhibitor exist in the sample, the absorbance of the sample tube will lower than the control tube. If the sample contains superoxide anion radical, the absorbance of the sample tube is higher than control tube. The ability of superoxide anion free radical in the sample can be calculated by using vitamin C as the standard.
Experimental instruments
Test tube, micropipettor, Vortex mixer, 37℃ water bath and spectrophotometer(550 nm)
Sample preparation
1. Serum (plasma): detect directly.
2. Tissue: Weigh the tissue accurately. Add normal saline in a weight (g): volume (mL) ratio of 1: 9, homogenize mechanically in ice water bath to break cells fully. Then centrifuge at 2500 rpm for 10 min at 4℃ and collect the supernatant for measurement. Meanwhile, determine the protein concentration of supernatant (E-BC-K318, E-BC-K168, E-BC-K165).
Operation steps
| Control tube | Standard tube | Sample tube |
R1 working solution (mL) | 1.0 | 1.0 | 1.0 |
ddH2O (mL) | 0.05 |
|
|
0.15 mg/mL Vc standard working solution (mL) |
| 0.05 |
|
Sample (mL) |
|
| 0.05 |
Reagent 2 (mL) | 0.1 | 0.1 | 0.1 |
Reagent 3 (mL) | 0.1 | 0.1 | 0.1 |
Reagent 4 working solution (mL) | 0.1 | 0.1 | 0.1 |
Mix thoroughly and incubate in 37℃ water bath for 40 min. | |||
Chromogenic reagent (mL) | 2.0 | 2.0 | 2.0 |
Mix thoroughly and stand for 10 min. Set the spectrophotometer to zero with ddH2O and measure the OD value at 550 nm with 1 cm optical path cuvette. | |||
Note: It is recommended to take a pre-experiment before formal experiment.
Note:
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 6 months.
4. Do not use components from different batches of kit.
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