Total Superoxide Dismutase (T-SOD) Activity Assay Kit (WST-8, Micro Method) - BioReagent

Cat. No.: T1505644

GRADE & PURITY BioReagent

Synonyms

Total Superoxide Dismutase Assay Kit with WST-8

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Application

Cell Metabolism, Enzyme activity assay

Overview

  The Total Superoxide Dismutase (T-SOD) Activity Assay Kit (WST-8, Micro Method) is a colorimetric kit for detecting SOD activity in cells, tissues, or other samples, based on a WST-8 chromogenic reaction. Superoxide Dismutase (SOD) catalyzes the dismutation of the superoxide anion (O₂•⁻) into hydrogen peroxide (H₂O₂) and oxygen (O₂), making it a crucial antioxidant enzyme in biological systems.

  While various methods exist for measuring SOD activity, the NBT (Nitro Blue Tetrazolium) method is widely used due to its convenience. However, the formazan dye produced by NBT has poor water solubility, can interact with reduced xanthine oxidase, and may not achieve 100% inhibition, affecting sensitivity and accuracy. The Cytochrome C method is another common technique, but cytochrome C has high oxidative activity, making it susceptible to interference from reducing agents in samples. Additionally, this method requires continuous absorbance monitoring, has relatively low sensitivity for SOD detection, and is less suitable for high-throughput sample analysis.

Assay Principle

Advanced methods for SOD determination include the WST-1 and WST-8 methods, with WST-8 being more stable and sensitive than WST-1. This kit employs the WST-8 method, known for its superior stability and high sensitivity, capable of detecting SOD activity as low as 0.5 U/mL. The principle of the WST-8 method is illustrated in Figure 1. WST-8 can react with the superoxide anion (O₂•⁻) generated by the xanthine oxidase (XO)-catalyzed reaction to produce a water-soluble formazan dye. Since SOD catalyzes the dismutation of the superoxide anion, this reaction step is inhibited by SOD. Therefore, SOD activity is negatively correlated with the amount of formazan dye produced. SOD enzyme activity can be calculated by colorimetric analysis of the WST-8 product.

Figure 1. Schematic diagram of the principle for detecting SOD enzyme activity based on the xanthine oxidase-coupled reaction system and WST-8.

Product Features

1. High Stability & Suitability for HTS: The WST-8 reaction product is a stable, water-soluble compound, allowing SOD activity determination by measuring absorbance at a single time point, making it ideal for high-throughput screening (HTS) research. The maximum inhibition percentage can approach 100%, and the assay is less affected by common interfering substances, significantly improving performance compared to other common methods.

2. Superior Performance: This kit outperforms similar Total SOD Activity Assay Kits based on the NBT method. It serves the same purpose but produces a significantly greater change in absorbance for an equivalent amount of SOD and offers a wider linear range.

3. Convenient Sample Preparation: The provided SOD Sample Preparation Solution directly lyses cells, eliminating the need for homogenization and simplifying operation.

4. Resistance to H₂O₂ Interference: Many cell and tissue samples contain endogenous hydrogen peroxide, which can interfere with SOD detection. This kit effectively removes interference from H₂O₂ in routine samples through the addition of appropriate amounts of catalase and other special methods. For example, adding up to 0.1 mM H₂O₂ to the SOD standard does not significantly affect the detection results.

5. Broad Applicability: This kit can detect SOD activity in various biological samples, including: cell or tissue homogenate supernatants, whole blood, red blood cell extracts, serum/plasma, pleural fluid, ascites, renal dialysate, urine, red blood cells, white blood cells, platelets, cultured myocardial cells, cultured tumor cells, and various animal/plant tissue cells and subcellular fractions.

T1505644	Component	100T	Storage
T1505644A	SOD Sample Preparation Solution	50 mL	2-8℃. Store in the dark.
T1505644B	SOD Assay Buffer	50 mL	2-8℃. Store in the dark.
T1505644C	WST-8	0.8 mL	2-8℃. Store in the dark.
T1505644D	Enzyme Solution	0.1 mL	2-8℃. Store in the dark.
T1505644E	Reaction Initiation Solution (40×)	0.06 mL	2-8℃. Store in the dark.

Experimental Procedure

1. Sample Preparation

1.1 Cell Sample Preparation

• Adherent Cells: Aspirate the culture medium. Wash cells once with ice-cold PBS or physiological saline. Add 100-200 µL of the provided SOD Sample Preparation Solution per 1 million cells. Pipette gently to lyse cells thoroughly.

• Suspension Cells: Collect cells by centrifugation at 600 g for 5 min. Wash the cell pellet once with ice-cold PBS or physiological saline. Add 100-200 µL of SOD Sample Preparation Solution per 1 million cells. Pipette gently to lyse cells thoroughly.

• Centrifuge all lysates at 12,000 g for 3-5 minutes at 4°C. Collect the supernatant as the test sample.

1.2 Tissue Sample Preparation

• Perfuse animals with physiological saline (0.9% NaCl containing 0.16 mg/ml heparin sodium) to remove blood before collecting tissue samples.

• Take an appropriate amount of tissue and homogenize it on ice using 100 µL of SOD Sample Preparation Solution per 10 mg tissue.

• Centrifuge the homogenate at 12,000 g for 3-5 minutes at 4°C. Collect the supernatant as the test sample.

1.3 Plasma or Red Blood Cell Sample Preparation

• Collect blood in an anticoagulant tube and mix gently by inverting.

• Centrifuge at 600 g for 10 min at 4°C. Transfer the supernatant (plasma) to a new tube. Dilute appropriately with physiological saline before assay.

• For red blood cell samples, refer to the method for suspension cells in section 1.1, or other sample preparation methods that do not contain detergents such as Triton X-100.

Notes:

• After preparation, determine the protein concentration using a BCA Protein Assay Kit. Typically, 10-20 µg of protein from cell or tissue homogenates contains approximately 1 unit of SOD activity (varies significantly). Preparing 20-100 µg of protein is usually sufficient.

• Dilute the sample appropriately with the provided SOD Assay Buffer based on the protein concentration and intended protein amount. For example, a 10% mouse liver homogenate supernatant usually requires a 10-100-fold dilution. Keep prepared samples on ice if assaying the same day; store at -70°C if not, but same-day assay is recommended.

2. Kit Reagent Preparation

2.1 WST-8/Enzyme Working Solution Preparation

• Prepare sufficient WST-8/Enzyme Working Solution for the number of assays (including standards), using 160 µL per reaction.

• For each 160 µL, mix 151 µL SOD Assay Buffer, 8 µL WST-8, and 1 µL Enzyme Solution uniformly.

• Refer to the table below for bulk preparation. Prepare fresh before use; keep on ice if stored temporarily during the day.

Number of Tests	1	10	20	50
SOD Assay Buffer (µL)	151	1510	3020	7550
WST-8 (μL)	8	80	160	400
Enzyme Solution (μL)	1	10	20	50
Total Volume (µL)	160	1600	3200	8000

Note: Centrifuge the Enzyme Solution vial briefly before use and mix gently due to its small volume and tendency to settle.

2.2 Reaction Initiation Working Solution Preparation

• Thaw and mix the Reaction Initiation Solution (40×). Dilute it 1:39 with SOD Assay Buffer (e.g., 1 µL reagent + 39 µL Buffer). Mix well.

• Prepare sufficient volume for all assays (including standards). Prepare fresh before use; keep on ice if stored temporarily during the day.

2.3 (Optional) SOD Standard Preparation

• Prepare an SOD standard yourself. Dilute it serially to the following concentrations using the SOD Sample Preparation Solution (if samples were prepared with it) or SOD Assay Buffer (for direct samples like blood): 100, 50, 20, 10, 5, 2, 1 U/mL.

• Use 20 µL per well in the assay as a reference. See Figure 2 for expected results.

• Note: Dilute the SOD standard immediately before use to prevent activity loss. The kit does not require an SOD standard for detection but can use it as a positive control or for quantitative reference.

3. Sample Assay

3.1 Plate Setup

• Set up sample wells and various blank control wells in a 96-well plate according to the table below.

• Add reagents in the specified order. Mix thoroughly after adding the Reaction Initiation Working Solution.

• Note: The reaction starts immediately after adding the Reaction Initiation Working Solution. Operate at low temperatures or use a multi-channel pipette to minimize timing errors between wells.

Reagent	Sample / Standard	Blank 1	Blank 2	Blank 3
Sample	20μl	/	/	20μl
SOD Assay Buffer	/	20μl	40μl	20μl
WST-8/Enzyme Working Solution	160μl	160μl	160μl	160μl
Reaction Initiation Working Solution	20μl	20μl	/	/

Note: Set up Blank 3 if the sample is colored or contains antioxidants. If not, Blank 3 is unnecessary.

3.2 Incubation

• Incubate at 37°C for 30 minutes. Incubation times between 25-35 minutes show no significant difference, but 30 minutes is recommended for consistency.

3.3 Absorbance Measurement

• Measure the absorbance at 450 nm. A filter in the range of 420-480 nm can be used.

• It is recommended to use a reference wavelength (e.g., 600 nm or 650 nm). Subtract the reference wavelength absorbance reading from the 450 nm reading for the final measured value (ΔA).

4. Calculation of Total SOD Activity

4.1 Calculation of Inhibition Percentage

Calculate the inhibition percentage using the following formula:

Inhibition Percentage (%) = [(A_Blank1 - A_Blank2) - (A_Sample - A_Blank3)] / (A_Blank1 - A_Blank2) × 100% 

If the sample is colorless and contains no antioxidants, A_Blank2 ≈ A_Blank3, and the formula simplifies to (Blank 3 can be omitted): Inhibition Percentage (%) = (A_Blank1 - A_Sample) / (A_Blank1 - A_Blank2) × 100%

Note: If the calculated inhibition percentage is <30% or >70%, re-assay the sample after appropriate dilution (if % is high) or concentration (if % is low). Aim for 30-70% inhibition for accurate results.

4.2 Definition of SOD Enzyme Activity Unit

One unit (U) of SOD activity is defined as the amount of enzyme that causes 50% inhibition in the described xanthine oxidase-coupled reaction system.

Note: SOD activity units can be defined in various ways; different units require appropriate conversion based on their definitions.

4.3 Calculation of SOD Enzyme Activity

Figure 2. Detection performance of this kit for SOD standards.

Figure A: Y-axis ΔA₄₅₀ is the absorbance difference between Blank 1 and the SOD standard well after 30 min incubation. 

Figure B: SOD enzyme activity has a nonlinear relationship with both ΔA₄₅₀ and Inhibition Percentage. 

Figure C: 1/SOD enzyme activity has a linear relationship with 1/Inhibition Percentage. 

Note: Data are for reference only; actual standard curve parameters may vary.

The SOD enzyme activity in the test sample can be calculated using the following formula:

• SOD Activity in Test Sample (Units) = Inhibition Percentage / (1 - Inhibition Percentage)

Examples:

• 50% Inhibition: Activity = 50% / (1 - 50%) = 1 Unit

• 60% Inhibition: Activity = 60% / (1 - 60%) = 1.5 Units

4.4 Normalization

• For cell or tissue homogenates, normalize the SOD activity units to U/mg protein or U/g tissue based on the sample's protein concentration and dilution factor.

• For red blood cell extracts, normalize to U/g hemoglobin or U/mg hemoglobin based on the hemoglobin content.

Appendix 1: Alternative Calculation Method (Reference)

Plot an inhibition percentage curve using the SOD standard. Calculate the sample SOD activity by comparing the sample's inhibition percentage to this curve. This method requires a reliable SOD standard and is not mandatory for using this kit.

Appendix 2: Kinetic Assay of SOD Activity

For increased precision, perform a kinetic read: after step 3.1, incubate at 37°C and continuously measure the absorbance at 450 nm for 30 minutes. Calculate the inhibition percentage using the slopes (rates) of absorbance change:

Inhibition Percentage (%) = [(Slope_Blank1 - Slope_Blank2) - (Slope_Sample - Slope_Blank3)] / (Slope_Blank1 - Slope_Blank2) × 100%

Subsequent calculations remain the same. The kinetic method is more precise but more complex.

Precautions

This product is for research use only. Not for use in diagnostic procedures.

Specifications

Synonyms

Total Superoxide Dismutase Assay Kit with WST-8

Specifications & Purity

BioReagent

Stability And Storage

Store at 2-8℃ long term (12 months). Store in the dark.

Storage

Store at 2-8°C, Protected from light

Shipped In

Wet ice

This product requires cold chain shipping. Ground and other economy services are not available.

Grade

BioReagent

Chemical and Physical Properties

• Sensitivity: Light-sensitive