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CuZn/Mn Superoxide Dismutase (CuZn-SOD/Mn-SOD) Activity Assay Kit (Hydroxylamine Method)
SKU: E-BC-K022-M-96
CuZn/Mn Superoxide Dismutase (CuZn-SOD/Mn-SOD) Activity Assay Kit (Hydroxylamine Method)
| SKU # | E-BC-K022-M |
| Detection Instrument | Microplate reader (530-570 nm, optimum wavelength: 550 nm) |
| Detection Method | Colorimetric method |
Product Details
Properties
| Synonyms | CuZn-SOD,Mn-SOD,T-SOD |
| Sample Type | Serum, plasma, urine, cells, cell culture supernatant, tissue |
| Sensitivity | 1.35 U/mL |
| Detection Range | 1.35-62 U/mL |
| Detection Method | Colorimetric method |
| Assay type | Enzyme Activity |
| Assay time | 70 min |
| Precision | Average inter-assay CV: 9.600% | Average intra-assay CV: 5.100% |
| Other instruments required | Micropipettor, Centrifuge, Incubator, Vortex mixer |
| Other reagents required | 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 15%~55% (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 |
| Mouse serum | 05-10 |
| Rat serum | 6-15 |
| Urine | 2-3 |
| Human hydrothorax | 2-3 |
| 10% Mouse liver tissue homogenate | 100-200 |
| 10% Mouse brain tissue homogenate | 20-30 |
| 10% Mouse kidney tissue homogenate | 50-120 |
| 10% Rat kidney tissue homogenate | 50-120 |
| HepG2 cells (5.21mgprot/mL) | 15-25 |
| Cell culture supernatant | 1 |
Note: The diluent is normal saline (0.9% NaCl).
Detection Principle
Superoxide anion (O2•-) produced by xanthine and xanthine oxidase system can oxidize hydroxylamine to form nitrite which appear purplish red after chromogenic reaction. The SOD in the sample has a specific inhibitory effect on superoxide anion (O2•-), can reduce the content of nitrite. The OD value is lower than control and the activity of SOD is calculated through the formula. There are two kinds of SOD (CuZn-SOD, Mn-SOD) in the cells of higher animals, and the sum of them is equal to the total SOD. The activity of Mn-SOD in the sample will lost after sample pretreatment, but the activity of CuZn-SOD will not.
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 via | 0.06 mL × 1 vial | -20℃, 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 |
| Reagent 9 | Extracting Solution | 6 mL × 1 vial | 12 mL × 1 vial | 2-8℃, 12 months shading light |
| 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) | 2.60 | 29.50 | 48.20 |
| %CV | 5.6 | 4.9 | 4.8 |
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) | 2.60 | 29.50 | 48.20 |
| %CV | 10.2 | 9.2 | 9.4 |
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 99%
| Sample 1 | Sample 2 | Sample 3 | |
| Expected Conc. (U/mL) | 13 | 34 | 55 |
| Observed Conc. (U/mL) | 13.1 | 33.3 | 53.9 |
| Recovery rate (%) | 101 | 98 | 98 |
Sensitivity
The analytical sensitivity of the assay is 1.35 U/mL. 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.