Total Superoxide Dismutase (T-SOD) Activity Assay Kit (WST-1 Method)

K141

96T(92 samples)

0.2 U/mL

0.2-14.4 U/mL

This kit is suitable for detecting the SOD activity in serum, plasma, pleural fluid, ascitic fluid, urine, cells and animal/plant tissues.

This kit can be stored at 2–8°C or –20°C in the dark for up to 12 months. Please refer to the manual for specific storage conditions of each reagent component.

WST-1 can react with superoxide anions (O2•−) to produce water-soluble formazan dye. Since SOD can catalyze the disproportionation reaction of superoxide anions, this reaction step can be inhibited by SOD. Therefore, the activity of SOD is negatively correlated with the amount of formazan dye produced. The activity of SOD can be calculated by colorimetric analysis of the WST-1 product.

When using this kit to detect tissue and cell samples, the total protein concentration needs to be determined. The BCA method (item number: K001) is recommended.

Instruments: Microplate reader (440-460 nm, optimum wavelength: 450 nm), Vortex mixer, micro pipette (1000 μL, 200 μL, 100 μL, 10 μL), multi-channel pipette (300 μL), 37°C incubator.

Consumables：pipette tip（1000 µL，200 µL，10 μL）、EP tube（10 mL，2 mL）。

Reagents: Double distilled water, Normal saline (0.9% NaCl)or PBS(0.01 M，pH 7.4)

① Take reagent 3 out from -20°C and place it on ice to slowly melt (it is best to aliquot it to avoid repeated freezing and thawing), while other reagents should be equilibrated to room temperature.

② Preparation of the enzyme working solution: Prepare it on an ice box. Mix reagent 3 and reagent 4 in a volume ratio of 1:10. The unused reagents can be stored at 2-8°C for 3 days.

③ Preparation of the substrate application solution: Mix reagent 2 and reagent 1 in a volume ratio of 1:200. The unused reagents can be stored at 2-8°C for 7 days.

① Sample processing

Sample requirements: The sample must not contain detergents such as SDS, Tween20, NP-40, Triton X-100, and reducing agents such as DTT, 2-mercaptoethanol.

Liquid samples (serum, plasma): After dilution at an appropriate ratio, directly measure.

Tissue samples: Homogenize according to the ratio of tissue sample mass (g): PBS (0.01 M, pH 7.4) (mL) = 1:9 (for example, 0.05 g tissue sample, add 450 μL PBS (0.01 M, pH 7.4) for homogenization), centrifuge at 4°C, 10000 × g for 10 minutes, take the supernatant and place it on ice for measurement. Take some of the supernatant for protein concentration measurement.

Cell samples: Take 10^6 cells and add 300-500 μL PBS (0.01 M, pH 7.4) for homogenization. After homogenization, centrifuge at 4°C, 10000 × g for 10 minutes, take the supernatant and place it on ice for measurement. Take some of the supernatant for protein concentration measurement.

② Sample dilution

This kit can detect SOD inhibition rates ranging from 25% to 65%, with the optimal inhibition rate range being 40% to 60%. Before the formal test, select 2-3 samples with significant differences and dilute them into different concentrations for pre-experiments. Select the dilution ratio within the optimal inhibition rate range for the formal batch experiment. The dilution ratio ranges for different samples are as follows (for reference only).

Note: The dilution solution is normal saline or PBS (0.01 M, pH 7.4).

If the SOD inhibition rate is greater than 65%, the sample needs to be diluted or the sampling volume reduced before testing; if the SOD inhibition rate is less than 25%, the sampling volume needs to be increased before testing.

Definition: In this reaction system, the enzyme quantity corresponding to a 50% inhibition rate of SOD is defined as one SOD activity unit (U).

Formula for calculating SOD inhibition rate:

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

The calculation formula for SOD activity in liquid samples such as serum (plasma):

SOD activity(U/mL) = i÷50%×V1÷V2×f

Formula for calculating the activity of SOD in tissues and cells:

SOD U/mgprot) = i÷50%×V1÷V2×f÷Cpr

Note:

ΔA1: OD value of Control well - OD value of Blank control well

ΔA2: OD value of Test well - OD value of Blank control well

i: SOD inhibition rate (%)

V1: Total volume of reaction solution (240 μL)

V2: Volume of sample added (20 μL)

f: Dilution factor of sample before adding to detection system

Cpr: Protein concentration of sample (mgprot/mL)

1. The kit is for research purposes only. If it is used for clinical diagnosis or any other purposes, our company will not be responsible for any issues arising therefrom, nor will it bear any legal liability.

2. Please read the manual carefully before the experiment and adjust the instrument properly. Conduct the experiment strictly in accordance with the manual.

3. During the experiment, please wear a lab coat and latex gloves to ensure protection.

4. The detection range of the kit is not equivalent to the concentration range of the analyte in the sample. If the concentration of the analyte in the sample is too high or too low, please appropriately dilute or concentrate the sample.

5. If the sample to be tested is not among the sample types listed in the manual, it is recommended to conduct a preliminary experiment to verify the detection validity.

6. The final experimental results are closely related to the effectiveness of the reagent, the relevant operations of the experimenter, and the experimental environment. Our company only takes responsibility for the reagent kit itself and does not bear any responsibility for the sample consumption caused by using the reagent kit. Please fully consider the possible usage amount of the sample before use and reserve sufficient samples.