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Total Superoxide Dismutase (T-SOD) Activity Assay Kit (Hydroxylamine Method)
SKU: E-BC-K019-M-96
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Total Superoxide Dismutase (T-SOD) Activity Assay Kit (Hydroxylamine Method)
| SKU # | E-BC-K019-M |
| Detection Instrument | Microplate reader (540-560 nm, optimum wavelength: 550 nm) |
| Detection Method | Colorimetric method |
Product Details
Properties
| Synonyms | T-SOD |
| Sample Type | Serum, plasma, urine, cells, cell culture supernatant, tissue |
| Sensitivity | 2.4 U/mL |
| Detection Range | 2.4-61 U/mL |
| Detection Method | Colorimetric method |
| Assay type | Enzyme Activity |
| Assay time | 70 min |
| Precision | Average inter-assay CV: 5.600% | Average intra-assay CV: 5.500% |
| Other instruments required | Micropipettor, Centrifuge, Incubator |
| Other reagents required | PBS (0.01 M, pH 7.4), Normal saline (0.9% NaCl) |
| Storage | Reagent 4: -20℃, others: 2-8℃ |
| Valid period | 12 months |
Dilution of sample
The optimal sampling volume are different for different species, the SOD also are different for different samples. It is recommended to take 2~3 samples to do a pre-experiment, diluting a series of diluent and determine the dilution factor when the SOD inhibition ratio is 10%~50% (the optimal inhibition ratio is the range of 25%~45%.) before formal experiment.
The recommended dilution factor for different samples is as follows (for reference only):
| Sample type | Dilution factor |
| Human serum | 2-4 |
| Rat serum | 4-6 |
| Mouse serum | 4-6 |
| HepG2 cell | 15-30 |
| Human urine | 2-5 |
| 10% Mouse liver tissue homogenate | 160-200 |
| 10% Epipremnum aureumtissue homogenate | 3-5 |
| 10% Mouse brain tissue homogenate | 80-100 |
Note: The diluent is normal saline (0.9% NaCl) or PBS (0.01 M, pH 7.4).
Detection Principle
The superoxide anion free radical (O2-) can be produced by xanthine and xanthine oxidase reaction system, O2- oxidize hydroxylamine to form nitrite, it turn to purple under the reaction of developer. When the measured samples containing SOD, the SOD can specifically inhibit superoxide anion free radical (O2-). The inhibitory effect of SOD can reduce the formation of nitrite, the absorbance value of sample tube is lower than control tube. Calculate the SOD of sample according to the computational formula.
Kit Components & Storage
| Item | Component | Size 1(48 T) | Size 2(96 T) | Storage |
| Reagent 1 | Buffer Solution | 1.2 mL × 1 vial | 1.2 mL × 1 vial | 2-8℃, 12 months |
| Reagent 2 | Nitrosogenic Agent | 1.2 mL × 1 vial | 1.2 mL × 1 vial | 2-8℃, 12 months |
| Reagent 3 | Substrate Solution | 1.2 mL × 1 vial | 1.2 mL × 1 vial | 2-8℃, 12 months |
| Reagent 4 | Enzyme Stock Solution | 0.03 mL × 1 vial | 0.06 mL × 1 vial | 2-8℃, 12 months |
| Reagent 5 | Enzyme Diluent | 1.2 mL × 1 vial | 1.2 mL × 1 vial | 2-8℃, 12 months |
| Reagent 6 | Chromogenic Agent A | Powder × 1 vial | Powder ×1 vial | 2-8℃, 12 months shading light |
| Reagent 7 | Chromogenic Agent B | Powder × 1 vial | Powder × 1 vial | 2-8℃, 12 months shading light |
| Reagent 8 | Chromogenic Agent C | 6 mL × 1 vial | 6 mL × 1 vial | 2-8℃, 12 months |
| Microplate | 96 wells | No requirement | ||
| Plate Sealer | 2 pieces | |||
Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents
Technical Data:
Parameter:
Intra-assay Precision
Three human serum samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (U/mL) | 3.50 | 25.00 | 45.00 |
| %CV | 5.9 | 5.4 | 5.2 |
Inter-assay Precision
Three human serum samples were assayed 20 times in duplicate by three operators to determine precision between assays.
| Parameters | Sample 1 | Sample 2 | Sample 3 |
| Mean (U/mL) | 3.50 | 25.00 | 45.00 |
| %CV | 5.5 | 5.3 | 6.0 |
Recovery
Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 105%.
| Sample 1 | Sample 2 | Sample 3 | |
| Expected Conc. (U/mL) | 15 | 34 | 55 |
| Observed Conc. (U/mL) | 16.1 | 36.0 | 56.1 |
| Recovery rate (%) | 107 | 106 | 102 |
Sensitivity
The analytical sensitivity of the assay is 2.4 U/mL T-SOD. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.