Total Superoxide Dismutase (SOD) Activity Colorimetric Assay Kit (WST-8 Method)
MCE Total Superoxide Dismutase (SOD) Activity Colorimetric Assay Kit (WST-8 Method) employs a water-soluble tetrazolium salt (WST-8)–based colorimetric system and quantifies total SOD activity in various biological samples by measuring the inhibition of WST-8 reduction to water-soluble formazan in the xanthine/xanthine oxidase system at 450 nm.
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Storage :
Reaction Stock Solution (40×), Enzyme Solution, SOD Sample Preparation Buffer, SOD Assay Buffer: 4°C, 1 year. Keep away from light. WST-8: -20°C, 1 year. Keep away from light.
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Description & Advantages
MCE Total Superoxide Dismutase (SOD) Activity Colorimetric Assay Kit (WST-8 Method) is based on a water-soluble tetrazolium salt (WST-8) chromogenic system (see schematic illustration of the assay principle). Superoxide anions (O2•-) generated by the xanthine–xanthine oxidase (XO) reaction system reduce WST-8 to a stable, water-soluble formazan product with a characteristic strong absorbance at 450 nm. SOD in the sample scavenges superoxide anions, thereby inhibiting the reduction of WST-8 to formazan and resulting in decreased color development. The color intensity is inversely correlated with SOD activity, i.e., higher absorbance indicates lower SOD activity and vice versa. Total SOD activity in samples can be quantitatively determined by measuring the absorbance at 450 nm using a visible spectrophotometer or microplate reader.
The reaction product of WST-8 is a stable, water-soluble formazan, allowing quantitative analysis based on a single time-point absorbance reading. This makes the assay well suited for high-throughput applications and drug screening. Compared with the conventional NBT method, the WST-8 method provides a broader inhibition response range for SOD activity, with a maximal inhibition rate approaching 100%, improved tolerance to common interfering substances, larger absorbance changes at equivalent SOD activities, a wider linear range, higher analytical sensitivity and can detect SOD as low as 0.5 U/mL.
This kit is suitable for the determination of SOD activity in a wide range of biological samples, including cell or tissue homogenate supernatants, whole blood, erythrocyte extracts, and serum. The provided SOD Sample Preparation Buffer enables direct lysis of cells without the need for additional homogenization steps, facilitating convenient and efficient sample preparation.
The kit is designed to eliminate interference from hydrogen peroxide and effectively minimizes the impact of endogenous H2O2 present in biological samples on SOD activity measurements. For example, the addition of up to 0.1 mM hydrogen peroxide to SOD standards does not significantly affect the assay results. The 100 T format is sufficient for 100 tests.
Protocol
Reagent Preparation
1. WST-8 working solution: prepare 160 μL per reaction by mixing 8 μL WST-8 with 152 μL SOD Assay Buffer. Prepare immediately before use and protect from light.
2. Enzyme working solution: for each reaction, mix 0.6 μL Reaction Stock Solution (40×), 1 μL Enzyme Solution, and 18.4 μL SOD Assay Buffer. Mix gently and prepare immediately before use.
Sample Preparation
2. Cells may be lysed directly with SOD Sample Preparation Buffer. For tissue, add approximately 100 μL SOD Sample Preparation Buffer per 10 mg tissue and homogenize on ice. Centrifuge at 12,000 × g for 10 min at 4°C and collect the supernatant.
3. Prepare whole-blood or erythrocyte lysates using a conventional method. Serum or plasma may be assayed directly or diluted with SOD Assay Buffer. Avoid repeated freeze-thaw cycles.
Assay Procedure
| Well | Sample/Buffer | Chromogenic Working Solution | Enzyme Working Solution/Buffer |
|---|---|---|---|
| Sample | 20 μL sample | 160 μL WST-8 working solution | 20 μL enzyme working solution |
| Blank Control 1 | 20 μL SOD Assay Buffer | 160 μL WST-8 working solution | 20 μL enzyme working solution |
| Blank Control 2 | 20 μL SOD Assay Buffer | 160 μL WST-8 working solution | 20 μL SOD Assay Buffer |
| Blank Control 3 | 20 μL sample | 160 μL WST-8 working solution | 20 μL SOD Assay Buffer |
1. Add samples or buffer and the chromogenic working solution to each well according to the table and mix gently.
2. Add 20 μL enzyme working solution to Sample and Blank Control 1 wells. Add 20 μL SOD Assay Buffer to Blank Control 2 and Blank Control 3 wells.
3. Mix gently and incubate at 37°C for 30 min protected from light.
4. Measure absorbance at 450 nm using a microplate reader.
Calculation
First calculate corrected absorbance: Asample = Sample well - Blank Control 3; Acontrol = Blank Control 1 - Blank Control 2.
SOD inhibition (%) = [Acontrol - Asample] ÷ Acontrol × 100%. One unit of SOD activity is defined as the amount of enzyme required to inhibit the chromogenic reaction by 50% under the assay conditions. Calculate SOD activity from the inhibition rate, sample volume, and dilution factor. The inhibition rate should be within 20%-80%; adjust sample dilution if it falls outside this range.
Storage
Reaction Stock Solution (40×), Enzyme Solution, SOD Sample Preparation Buffer, SOD Assay Buffer: 4°C, 1 year. Keep away from light.
WST-8: -20°C, 1 year. Keep away from light.
Attention
1. Test samples may be stored at -70°C for up to 1 month. Repeated freeze/thaw cycles should be strictly avoided, as they may cause partial inactivation of SOD activity and compromise the accuracy of the assay results.
2. During the preparation of cell or tissue samples, lysis buffers or extraction solutions containing detergents such as Triton X-100 should be avoided, as they may interfere with the reaction system of this kit and affect the reliability of the assay results.
3. Antioxidants may interfere with this assay. For example, 0.1 mM ascorbic acid or 5 mM reduced glutathione (GSH) can significantly increase the measured absorbance. In such cases, even if the samples themselves are colorless, setting up Blank Control 3 as described in the protocol can effectively eliminate the interference caused by antioxidants in the samples.
4. This product is for R&D use only, not for drug, household, or other uses.
5. For your safety and health, please wear a lab coat and disposable gloves to operate.
Components
| Cat. No. | Product List | Components | HY-K0325-100 T | Storage |
|---|---|---|---|---|
| HY-K0325-A | Assay Reagent A | Reaction Stock Solution (40×) | 60 μL | 4°C, 1 year. Keep away from light. |
| Enzyme Solution | 100 μL | |||
| SOD Sample Preparation Buffer | 50 mL | |||
| SOD Assay Buffer | 50 mL | |||
| HY-K0325-B | Assay Reagent B | WST-8 | 800 μL | -20°C, 1 year. Keep away from light. |
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