Total Superoxide Dismutase Assay Kit with NBT - BioReagent, high purity

Cat. No.: T1373303

GRADE & PURITY BioReagent

Overview

Superoxide Dismutase (SOD) is a metalloenzyme that catalyzes the dismutation of superoxide anion into hydrogen peroxide (H₂O₂) and oxygen (O₂). It is an essential antioxidant enzyme in organisms. Since superoxide radicals are unstable and have an extremely short lifespan, the activity of SOD is generally determined by indirect methods combined with various color reactions. Common chromogenic agents include Nitro Blue Tetrazolium (NBT), WST-1 and WST-8.

This product adopts the NBT-based photoreduction principle, also known as the NBT photoreduction method. The detection mechanism is as follows: riboflavin undergoes photoreduction in the presence of oxidizing substances. Under aerobic conditions, the reduced riboflavin is readily re-oxidized to generate superoxide anion radicals. These radicals reduce NBT into blue formazan, which has a strong absorbance at 560 nm. SOD scavenges superoxide anions and thus inhibits the formation of formazan.After the photoreduction reaction, the darker the blue color of the reaction solution, the lower the SOD activity; conversely, the higher the enzyme activity. The total SOD activity in samples can be calculated via colorimetric analysis using a microplate reader. This method quantifies SOD activity by measuring its inhibitory effect on the light-induced reduction of NBT. It is applicable to detect SOD activity in plants, tissues, cells, serum and other specimens. This kit is intended for research use only and is not for clinical diagnosis or other applications.

Product Components

T1373303	Component	100 T	Storage
T1373303A	SOD Extraction Reagent	2×250ml	RT
T1373303B	Met buffer solution	20ml	2-8 ℃. Store in the dark.
T1373303C	NBT solution	3ml	2-8 ℃. Store in the dark.
T1373303D	FD Stock Solution	3ml	2-8 ℃. Store in the dark.

Materials to be Prepared by User

1. Normal saline or PBS, deionized water.

2. Electronic balance, scissors, low-temperature refrigerator or ice maker, ice packs, homogenizer or mortar, centrifuge, centrifuge tubes, test tubes, 40W fluorescent lamp, illuminance meter, microplate reader, 96-well plate.

Operating Procedures (For Reference Only)

1. Sample Preparation

① Plasma or erythrocyte-containing samples

The separated serum or plasma shall be free of hemolysis. If hemolysis occurs, remove erythrocytes before detection. If the sample value exceeds the detection range, dilute it with SOD extraction reagent prior to testing.

Simple method for removing erythrocytes from serum: Collect blood using an anticoagulant tube and mix by inverting. Take no less than 500 μL whole blood, centrifuge at 3000 rpm for 5 minutes at 4 °C. Transfer the supernatant to a new 1 mL centrifuge tube, dilute with an appropriate amount of normal saline for later detection. Erythrocyte lysis solution (e.g., ACK lysis buffer) can also be used to remove erythrocytes.

② Tissue samples

Perfuse animal tissues with 0.9% NaCl normal saline containing 20 U/mL Heparin to eliminate residual blood before sampling. Add 500 μL SOD extraction reagent per 100 mg tissue. Homogenize the sample with a glass homogenizer on ice or at 4 °C. Centrifuge the homogenate at 4000 rpm for 10 minutes at 4 °C, and collect the supernatant (crude SOD extract) for activity assay.

③ Cell samples

For adherent cells, avoid trypsin digestion to preserve enzyme activity. Harvest cells using a cell scraper or EDTA treatment. Rinse cells once with sterile PBS or normal saline. Add 300–500 μL SOD extraction reagent per 10⁶ cells, and homogenize with a glass homogenizer on ice or at 4 °C. Centrifuge at 10000 rpm for 10 minutes at 4 °C, then collect the supernatant (crude SOD extract) for activity assay.

④ Plant samples

Accurately weigh 0.4 g plant material (pulp or leaf blades with veins removed) and cut into pieces. Place the sample in a pre-chilled mortar or homogenizer at 4 °C, add 1 mL pre-cooled SOD extraction reagent, and grind thoroughly on ice. Transfer the homogenate to a centrifuge tube. Rinse the mortar or homogenizer with 3 mL SOD extraction reagent and combine the rinse solution into the same tube. Top up with extraction reagent to a total volume of 4 mL. Centrifuge at 4000 rpm for 20 minutes at 4 °C; the resulting supernatant is the enzyme extract for SOD detection.

Note: If the SOD activity is low, reduce the total volume of extraction reagent to increase enzyme concentration.

⑤ Post-treatment of prepared samples

Determine the protein concentration of samples via the BCA method. Generally, 10–20 μg protein from cell or tissue homogenate contains approximately 1 unit of SOD activity (values vary greatly across cell and tissue types, for reference only). A protein content of 20–100 μg per sample is sufficient for subsequent detection.

Dilute samples appropriately with the supplied SOD extraction reagent according to protein concentration and required dosage. For example, 10% mouse liver tissue homogenate (weight ratio of tissue to homogenate: 10%) usually requires a dilution ratio of 10–100 times. Store prepared samples on ice if assayed on the same day. For delayed detection, freeze samples at -20 °C. Assay samples on the day of preparation whenever possible.

2. (Optional) Preparation of SOD Standard

Prepare a series of SOD standard solutions at concentrations of 200, 100, 50, 20, 10, 5 and 2 U/mL using the supplied SOD extraction reagent. Pipette 20 μL of each standard solution for detection.

Note: Prepare and use the standard solutions immediately to prevent activity loss. SOD standard is not mandatory for this assay, but it can serve as a positive control or reference for quantitative analysis.

3. Selection of Light Source

Use an illuminance meter to identify positions with a light intensity of 3500–4000 Lux in a light incubator or under fluorescent lamps for the photoreaction, and mark the positions.

4. Preparation of NBT Working Solution

Mix Met buffer and NBT solution at a volume ratio of 23:3.

5. Preparation of FD Working Solution

Mix FD stock solution with deionized water at a volume ratio of 2:1 to 2:3. The solution is ready for use when its absorbance at 466 nm reaches 0.30–0.45. Adjust the volume of deionized water to bring the absorbance into the target range if needed.

6. Sample Loading for SOD Assay

Set up blank control wells, light control wells and test wells in a 96-well plate as specified in the attached table. Add test samples and other reagents sequentially under low light intensity, and mix thoroughly after adding FD working solution.

Note: The reaction starts immediately after FD working solution is added. Use a multi-channel pipette under low light to minimize errors caused by inconsistent reagent adding time across wells.

Additive (μL)	Blank control well	Light control well	Test well
SOD Extraction Reagent	8	8	-
Test sample (supernatant)	-	-	8
NBT working solution	208	208	208
FD working solution	24	24	24

7. SOD Measurement

Mix the solutions thoroughly. Place the blank control wells in the dark, and incubate all other wells under a 4000 Lux fluorescent lamp for 20 minutes. Ensure uniform light exposure for all wells. Adjust the reaction duration: shorten it at high temperatures and extend it at low temperatures. After the reaction, immediately set the blank control wells (without light exposure) as the zero reference, then measure the absorbance at 560 nm using a microplate reader.

Calculation 

Definition of SOD Activity Unit: One unit (U) of enzyme activity is defined as the amount of SOD causing 50% inhibition of the photochemical reduction of NBT.

Total SOD activity in liquid (U/mL)=(A_{light control}−A_test) ×V₁/ (50%×A_{light control}×V_T)       

Total SOD activity in tissue and cell homogenate (U/g)=(A_{light control}−A_test)×V₁×V/ (50%×A_{light control}×V_T×W)   

Parameter explanation

    ·  A_{light control}: Absorbance of light control well

    ·  A_test: Absorbance of test well

    ·  V₁: Total volume of reaction solution (mL)

    ·  V: Total volume of sample solution (mL)

    ·  V_T: Volume of sample used in detection (mL)

    ·  W: Fresh weight of sample (g)

    ·  C: 1 mL/Volume of blood collected (mL)

Precautions

1. Do not repeatedly freeze and thaw low-temperature reagents, as this may cause reagent failure or reduced performance. Test samples can be stored at -70 °C for one month. Repeated freeze-thaw cycles will partially inactivate SOD.

2. Polyphenols in plant samples can trigger irreversible precipitation of enzyme proteins and lead to enzyme deactivation. Therefore, polyphenol adsorbents must be added during SOD extraction to remove such substances and prevent protein denaturation. The supplied SOD extraction reagent contains PVP and other components to effectively eliminate polyphenols. If the extraction reagent is insufficient, 0.05 M sodium phosphate buffer (pH 7.8) can be used as a substitute.

3. Avoid solutions containing detergents such as Triton X-100 during the preparation of cell and tissue samples, which will interfere with the assay results.

4. Antioxidants may interfere with the detection. For instance, 0.1 mM ascorbic acid and 5 mM GSH will significantly increase the measured absorbance values.

5. Grind plant samples quickly on ice to prevent the decline of SOD activity.

6. If a spectrophotometer is used, cuvettes with a 1 cm optical path length are required. The volumes of supernatant and reagents added shall comply with the minimum volume requirement of the cuvettes.

7. Use clean, transparent centrifuge tubes or 96-well plates with good light transmittance.

8. Ensure uniform light exposure for all wells. Arrange all reaction vessels in a line parallel to the fluorescent lamp tube.

9. Maintain the reaction temperature at 25 °C, and adjust the reaction time according to SOD activity. Shorten the illumination time at higher temperatures and extend it at lower temperatures.

10. If a 4000 Lux light source is unavailable, alternative light conditions are as follows: Use a 200 W lamp at a distance of 10–12 cm for 20 minutes; The light intensity is about 800–1000 Lux at 5 cm above a clean bench, with an illumination time of 30–40 minutes. Direct strong sunlight (approximately 30000 Lux) only requires 5–15 minutes of irradiation, which is not recommended.

11. Wear lab coats and disposable gloves during operation for personal safety and health.

12. Use opened reagents promptly to avoid affecting subsequent experiments.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature

Shipped In

Wet ice

Stability And Storage

T1373303A SOD extraction reagent Store at RT long term (12 months) T1373303B Met buffer Store at 2-8 ℃ long term (12 months). Store in the dark T1373303C NBT solution Store at 2-8 ℃ long term (12 months). Store in the dark T1373303D FD mother liquor St