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Inhibition And Production Of Superoxide Anionic Colorimetric Assay Kit(WST-1 Method)

Cat.No.:E-BC-K001-M
Detection instrument: Microplate reader(440-460 nm,optimum wavelength: 450 nm)

Detection method: Colorimetric method

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Detection principle

Superoxide anion free radicals are produced through the reaction system of xanthine and xanthine oxidase. WST-1 (a water-soluble tetrazolium salt) can react with the generated superoxide anion to produce water-soluble formazan. When the tested sample contains the superoxide anion free radical inhibitor, it can inhibit the formation of formazan. When the tested sample contains the substance that produces superoxide anion free radical, it can promote the formation of formazan dye. By colorimetric analysis of WST-1 products, the units of activity of inhibition or production of superoxide anion radical in samples can be calculated.

Performance characteristics

Sample type	Serum,plasma,urine,cells,cell culture supernatant,leucocyte
Detection method	Colorimetric method
Assay type	Activity
Assay time	35 min
Precision	Average inter-assay CV: 5.800%Average intra-assay CV: 2%
Other instruments required	Micropipettor, Multi-channel pipettor, Incubator, Vortex mixer, Centrifuge
Other reagents required	Normal saline (0.9% NaCl), PBS (0.01 M, pH 7.4)
Storage	Reagent 3: -20℃, others: 2-8℃
Valid period	12 months

Dilution of sample

Before the formal experiment, it needs to choose 2-3 samples for diluting a series of diluent and determine the dilution factor when the SOD inhibition ratio is 30%~65% (the optimal inhibition ratio is the range of 40%~60%).

Inhibition ratio=[(A1-A2)-(A5-A6)]×100%/(A1-A2)

Adjust sampling volume: If inhibition ratio > 65%, need to dilute the sample or decrease the sampling volume than take the test. If inhibition ratio < 30%, need to increase the sampling volume.

The recommended dilution factor for different samples is as follows (for reference only):

Sample type	Dilution factor
Human serum	4-7
Mouse serum	15-25
Rat serum	25-35
Human saliva	1
HepG2 culture supernatant	1
10% Rat brain tissue homogenate	150-200
10% Rat liver tissue homogenate	500-600
10% Mouse liver tissue homogenate	500-600
10% Mouse heart tissue homogenate	150-200
10% Epipremnum aureum tissue homogenate	20-30

Note: The diluent is normal saline (0.9% NaCl) or PBS (0.01 M, pH 7.4).

Troubleshooting

Collection and treatment of common samples for metabolism assay detection

Questions and Answers of Metabolism Assay Kits

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Q&A

R**eSubmitted [ Feb 03 2024 ]

Asked: Hallo, ive been used this assay .. i want to ask because there was some confused about the result. inhibition activity of superoxide radical, if the value are high, it means inhibition proecess is high? or the level production Superoxide that \"should\" be inhibit are high? Which one ?

adminSubmitted [ Feb 03 2024 ]

Answered: At first, you need to determine whether the sample can inhibit superoxide anion radical or produce superoxide radicals in the reaction, and then select the appropriate calculation formula for analysis. If your sample is to inhibit superoxide anion in the reaction, please choose formula 1 and 2 to calculate. The larger the measured value, the better the inhibition effect. If your sample produces superoxide anions in the reaction, then choose formula 3 to calculate.The larger the measured value is, the more superoxide anions are generated, and the better the generated effect is.

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