太鼎生物科技/太鼎食安科技

Superoxide Dismutase (SOD) Activity Assay Kit
Catalog#: K335-100  | Size: 100 assays

Kit Summary:

• Detection method- Absorbance (450 nm)
• Sample type- Cell and Tissue lysates, culture media, urine, plasma and serum, as well as many other biological fluids
• Species reactivity- Mammalian
• Applications- Kit contains the necessary reagents for convenient measurement of activity of Superoxide Dismutase (SOD) by colorimetric method.

Features & Benefits:
• Simple one-step procedure; takes around than 30 minutes
• Fast and convenient

Kit components:
• WST Solution
• SOD Enzyme Solution
• SOD Assay Buffer
• SOD Dilution Buffer

Description:
Superoxide dismutase (SOD) is one of the most important antioxidative enzymes. It catalyzes the dis mutation of the superoxide anion into hydrogen peroxide and molecular oxygen. The sensitive SOD assay kit utilizes WST-1 that produces a water-soluble formazan dye upon reduction with superoxide anion. The rate of the reduction with a superoxide anion is linearly related to the xanthine oxidase (XO) activity, and is inhibited by SOD (below). Therefore, the inhibition activity of SOD can be determined by a colorimetric method.

II. Reagent Preparation and Storage Conditions:

WST Working Solution: Dilute the 1 ml of WST solution with 19 ml of Assay Buffer Solution. The diluted solution is stable for up to 2 months at 4C .

Enzyme Working Solution: Centrifuge the Enzyme Solution for 5 seconds. Mix well by pipeting (The step is necessary, as the enzyme has two layers and must be mixed well before dilution). Dilute 15 l with 2.5 ml of Dilution Buffer. The diluted enzyme solution is stable for up to 3 weeks at 4C .

III. Sample Preparation:

1. Blood samples: Collect blood using citrate or EDTA. Centrifuge at 1,000 x g for 10 min at 4C . Transfer the plasma layer to a new tube without disturbing the buffy layer and store at -80°C until ready for analysis. Remove the buffy layer from the red cell pellet. Resuspend the erythrocytes in 5x volume of ice cold distilled water and centrifuge at 10,000 x g for 10 min to pellet the erythrocyte membranes. Store the supernatant at -80C until ready for analysis. Plasma can be diluted approx. 3-10x and the red cell lysate diluted approx. 100x prior to SOD assay.

2. Tissue and cells: Tissue should be perfused with PBS or 150mM KCl to remove any red blood cells. Homogenize tissue or lyse cells in ice cold 0.1M Tris/HCl, pH 7.4 containing 0.5 % Triton X-100, 5mM β-ME, 0.1mg/ml PMSF. Centrifuge the crude tissue homogenate/cell lysate at 14000 x g for 5 minutes at 4C and discard the cell debris. The supernatant contains total SOD activity from cytosolic and mitochondria.

 Fractionation Kit. SOD activity is then measured from the Mitochondria and Cytosol fractions separately.

 SOD Assay Protocol:

*Refer to Table 1 for the amount of solution in each well. If you are using a SOD standard (not included with the kit), set up wells for it in the same manner as the sample.

1. Add 20 l of Sample Solution to each sample and blank 2 well and add 20 l H2O to each Blank 1 and Blank 3 well (See Table I).

2. Add 200 l of the WST Working Solution to each well.

3. Add 20 l of Dilution Buffer to each Blank 2 and Blank 3 well.

4. Add 20 l of Enzyme Working solution to each sample and Blank 1 well, mix thoroughly.

Note: since the superoxide will release immediately after the addition of Enzyme working Solution to each well, use a multiple channel pipette to avoid reaction time lag of each well.

5. Incubate plates at 37C for 20 minutes.

6. Read the absorbance at 450 nm using a microplate reader.

7. Calculate the SOD activity (inhibition rate%) using the following equation.

If it is desired to measure SOD activity from cytosol and mitochondria separately, cytosol and Mitochondria can be separated by using BioVision K256-100 Mitochondrial/Cytoso

Storage Conditions:
+ 4°C

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