Total Superoxide Dismutase (T-SOD) Activity Assay Kit (for Biochemical Assay Kit)

Product Information

Product Description/Introduction

Superoxide dismutase (SOD), which catalyzes the disproportionation of superoxide anions to generate hydrogen peroxide and oxygen. SOD is an important antioxidant enzyme in living organisms and plays a crucial role in the oxidative and antioxidant balance of the organism.

This kit used WST-1 method to detect and analyze SOD activity. The principle is as follows: Xanthine produces superoxide anion under the action of xanthine oxidase, which can react with WST-1 to produce yellow water-soluble methyl dyes.SOD can catalyze the dismutation of superoxide anions and reduce the content of superoxide anions, that is, the activity of SOD is negatively correlated with the formation of methylate dyes.Therefore, the activity of SOD can be calculated by colorimetric analysis of WST-1.

Storage and Shipping Conditions

Ship with wet ice; PBS buffer to be stored at 2-8; other components to be stored at -20 away from light, valid for 6 months.

Product Content

Assay Protocol / Procedures

1. Sample Preparation:

1.1. Plasma and serum samples can be directly used for the detection of T-SOD activity;

1.2. Tissue samples: Use suitable buffer (PBS, saline, etc.) for homogenization and lysis, where the ratio of tissue to reagent is 1 to 9 (0.1 g : 0.9 mL); After homogenization and lysis, centrifuge at 4, 10,000 g for 10-15 min, and take the supernatant for the subsequent T-SOD viability assay;

1.3. Cell samples: After cell collection, cells can be resuspended with a suitable buffer solution, approximately 300-500 μL of PBS or saline per 1 X 106 cells; after homogenization, centrifuge the cells at 10,000 g for 10-15 min at 4°C, and remove the supernatant for subsequent T-SOD viability assay.  

Note: Try not to include surfactants such as SDS, Triton-X-100, Tween 20, and reducing agents such as reduced glutathione, DTT, and 2-mercaptoethanol in the sample.

2. Pre-test preparation:

2.1. SOD color development substrate: SOD assay buffer is mixed 1:100 to prepare SOD assay working solution, which is ready for use;

2.2. Blow up the enzyme stock solution before use and mix with SOD assay buffer 1 to 20 to formulate the enzyme working solution, ready to use;

3. T-SOD detection:

3.1. Conduct detection according to the following table:

Note: The reaction starts immediately after the assay working solution is added. It is recommended to use a multi-channel pipette to add the assay working solution to minimize experimental errors;

4. Data analysis:

Before data analysis, the protein concentration of the tissue or cell samples to be tested should be calculated. The protein concentration can be determined by BCA Protein Quantification Kit (G2026).

Definition: In this reaction system, the amount of enzyme corresponding to 50% inhibition of SOD is one SOD activity unit (U);

4.1. Result calculation:

4.1.1. T-SOD inhibition rate calculation formula:

4.1.2. T-SOD inhibition rate (%) =（ΔA1-ΔA2 ）÷ΔA1×100%

4.1.3. Formula for calculating T-SOD activity in liquid samples such as serum (plasma):

T-SOD activity (U/mL) = i÷（100%-i）×V1÷V2×f

4.1.4. Calculation formula of T-SOD activity in tissues and cells:

T-SOD activity (U/mgport) = i÷（100%-i）×V1÷V2÷×f

ΔA1 is the control OD value-control blank OD value; ΔA2 is the assay OD value-assay blank OD value; i is the T-SOD inhibition (%); V1 is the total volume of the reaction (0.24 mL); V2 is the volume of the sample (0.02 mL);  is the protein concentration of the sample (mgprot/mL); and f is the dilution.

Note

1. If the sample has color or contains a small amount of antioxidant substances, it is necessary to set up the determination blank wells; otherwise, it is not necessary to set up the determination blank wells. When analyzing the data and calculating the inhibition rate, the OD value of the control blank can be used to replace the OD value of the assay blank.

2. The inhibition rate of SOD detected by this kit should be in the range of 30-70%, and the inhibition rate of 40-60% is the best. Before the formal test, it is recommended to select several samples with large differences to dilute into different concentrations for pre-test to ensure that they are within the detection range.

3. If there are bubbles in the holes, blow them gently with the pipette to avoid affecting the test results.

4. For your health and safety, please wear lab coat and gloves during operation.

Appendix: Technical parameters

For Research Use Only!

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