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Total Superoxide Dismutase Activity Assay Kit (Hydroxylamine Method)– MSE Supplies LLC

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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.