CuZn/Mn Superoxide Dismutase (CuZn-SOD/Mn-SOD) Activity Assay Kit (Hydroxylamine Method)

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.