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Cat. No.: CZSMSAK-100 (for 100T)

Description

Cu/Zn-SOD and Mn-SOD Assay Kit is a kit based on the WST-8 colorimetric reaction, used to detect the activity of Cu/Zn-SOD, Mn-SOD, or total SOD in cells, tissues, or other samples through colorimetry.

Superoxide dismutase (SOD) catalyzes the dismutation of superoxide anions to produce hydrogen peroxide (H₂O₂) and oxygen (O₂), making it an important antioxidant enzyme in organisms.

Currently, there are various methods for measuring SOD activity. The NBT (Nitro Blue Tetrazolium) method is widely used due to its convenience. However, the formazan dye produced by the NBT method has poor water solubility and can interact with reduced xanthine oxidase, leading to less than 100% inhibition, which affects the sensitivity and accuracy of detection. The cytochrome C method is another common method for detecting SOD activity, but cytochrome C has high oxidative activity and is easily interfered with by reducing agents in the sample. Additionally, this method requires continuous measurement of absorbance values, making it less sensitive for SOD detection and not very suitable for large-scale sample testing.

The more advanced methods for measuring SOD include the WST-1 method and the WST-8 method, with the WST-8 method being more stable and sensitive than the WST-1 method. This kit uses the WST-8 method, which is more stable and sensitive among the current SOD measurement methods.

The reaction product of WST-8 is a stable, water-soluble product that allows for the measurement of SOD activity through absorbance detection at a single time point, making it suitable for high-throughput screening studies. When measuring SOD enzymatic activity using the WST-8 method, the maximum inhibition percentage can approach 100%, and it is not affected by some common interfering factors, significantly improving detection results compared to other common methods.

The detection of this kit is not affected by hydrogen peroxide in the sample. Many cell and tissue samples contain endogenous hydrogen peroxide, which can interfere with SOD detection. This kit effectively removes the interference of hydrogen peroxide in conventional samples by adding an appropriate amount of catalase and other special methods. For example, when detecting SOD standards, adding up to 0.1 mM hydrogen peroxide to the standard does not significantly affect the detection results.

Mammals encode three types of SOD. In most tissues, the highest content is Cu/Zn-SOD located in the cytoplasm. Mn-SOD is mainly expressed in mitochondria and can be inducibly expressed. Mn-SOD can also be located in the cytoplasm under certain conditions. Some tissues or body fluids contain a third type of SOD, extracellular SOD (EC-SOD). EC-SOD is also a type of Cu/Zn-SOD, highly similar to the gene encoding cytoplasmic Cu/Zn-SOD but with some differences. In summary, there are two types of SOD in cells or tissues: Cu/Zn-SOD and Mn-SOD. The total activity of these two SOD enzymes is the total SOD activity. When the enzyme activity of Cu/Zn-SOD is inhibited by its inhibitor, the measured activity is that of Mn-SOD. If the total SOD enzyme activity is measured simultaneously, the enzyme activity of Cu/Zn-SOD can be calculated.

KCN or NaCN can inhibit Cu/Zn-SOD enzyme activity and are widely used to measure Mn-SOD enzyme activity. However, both KCN and NaCN are highly toxic and do not achieve inhibition percentages close to 100% for Cu/Zn-SOD.

This kit uses a non-toxic two-step dual inhibition method for Cu/Zn-SOD, ensuring a high inhibition percentage close to 100% for Cu/Zn-SOD without using toxic substances. This makes the measurement of Cu/Zn-SOD or Mn-SOD enzyme activity safer and more accurate.

This kit can detect SOD activity in biological samples such as cell or tissue homogenate supernatants, whole blood, red blood cell extracts, and serum.

Components

• SOD Detection Buffer
• WST-8
• Enzyme Solution
• Reaction Initiator (40X)
• Cu/Zn-SOD Inhibitor A
• Cu/Zn-SOD Inhibitor B

Principle

WST-8 can react with superoxide anions produced by the catalysis of xanthine oxidase to generate a water-soluble formazan dye. This reaction step can be inhibited by SOD. By performing colorimetric analysis of the WST-8 product, the enzymatic activity of SOD can be calculated.

Storage

Store at -20°C, valid for six months. WST-8 should be stored protected from light.

Precautions

• Test samples can be stored at -70°C for one month. Note that repeated freeze-thaw cycles can lead to partial inactivation of SOD.
• When preparing samples such as cells or tissues, do not use solutions containing detergents like Triton X-100, as they will interfere with the detection of this kit.
• Antioxidants can interfere with the detection of this kit. For example, 0.1 mM ascorbic acid and 5 mM GSH can significantly increase the measured absorbance. Although the sample may not have color, setting blank control 3 as described in the instructions can eliminate the interference of antioxidants in the sample.
• Cu/Zn-SOD Inhibitor A is irritating to humans. Please handle with care and take appropriate precautions to avoid direct contact with the body or inhalation.
• This product is for scientific research use by professionals only and is not intended for clinical diagnosis or treatment, food, or drugs, and should not be stored in ordinary residences.
• For your safety and health, please wear a lab coat and disposable gloves during operation.