According to the literature, superoxide dismutase exists in all oxygen-metabolizing cells to protect cells from excessive superoxide. Under the action of SOD, two superoxide anions were converted to oxygen and hydrogen peroxide.
In mammals, there are three different forms of SOD: CuZn-SOD, Mn-SOD and EC-SOD (an extracellular form of SOD). Cu-Zn SOD exists in the cytoplasmic and mitochondrial membrane spaces of the cells, while Mn-SOD is located in the mitochondrial matrix.
1. Determine optimal sampling volume of each sample before formal experiment. Calculate the inhibition ratio of serial sampling volume, and choose the optimal sampling volume when inhibition ratio in the range of 25%~45%.
2. The optimal sampling volume are different for different species, the SOD also are different for different samples. So it is best to do a pre-test to determining optimal sampling volume for a new sample.
3. It is best to reserve 3 paralleled tubes with different sampling volumes in pre-test for determining the optimal sampling volume. The sampling volume in examples as median, increase by 10 μL and decrease by 10 μL. Take the pre-test with 3 paralleled tubes and 1 control tube to determining the optimal sampling volume.
4. Adjust sampling volume: If inhibition ratio > 55%, need to dilute the sample or decrease the sampling volume than take the test. If inhibition ratio < 15%, need to increase the sampling volume.
5. All the reagents should be prepared at the day before the experiment, in order to let the reagents dissolve fully. Please bring all the reagents and samples to room temperature for 30 min before the assay.
6. The incubation time is 40 min, the incubation time can be extended to 45 min when the room temperature is lower than 20℃. Ensure the incubation temperature is 37℃.
7. EDTA should not be as anticoagulation, suggest to use heparin plasma.
1. Sample tube: add 1 mL of Reagent 1 working solution and a* mL sample to the Sample tubes.
Control tube: add 1 mL of Reagent 1 working solution and a* mL double-distilled water to the Control tubes.
2. Add 0.1 mL of Reagent 2, 0.1 mL of Reagent3, 0.1 mL of Reagent 4 working solution successively into the tubes of Step 1.
3. Mix fully with a vortex mixer, incubate for 40 min at 37 ℃.
4. Add 2 mL of Chromogenic agent into the tubes of Step 3.
5. Mix fully and stand for 10 min at room temperature.
6. Set to zero with double-distilled water and measure the OD value of each tube at 550 nm with 1 cm optical path quartz cuvette.
[Note]: If the optimal sampling volume (a*) is the same, only one control tube need to be assay.Reagent | Sample tube | Control tube |
Reagent 1 working solution (mL) | 1.0 | 1.0 |
Sample (mL) | a* |
|
Double distilled water(mL) |
| a* |
Reagent 2 (mL) | 0.1 | 0.1 |
Reagent 3 (mL) | 0.1 | 0.1 |
Reagent 4 working solution (mL) | 0.1 | 0.1 |
Mix fully with a vortex instrument, incubate for 40 min at 37 ℃. | ||
Chromogenic agent | 2 | 2 |
Mix fully and stand for 10 min at room temperature. Set to zero with double-distilled water and measure the OD value of each tube at 550 nm with 1 cm diameter cuvette. | ||
| Detection range | 4.7-166 U/mL | Average inter-assay CV | 6.3% |
| Sensitivity | 4.7 U/mL | Average intra-assay CV | 2.8% |
| Average recovery rate | 105% |
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