Superoxide Dismutase (SOD) Assay Kit (NBT) - BioReagent, high purity

Cat. No.: T1373360

GRADE & PURITY BioReagent

Overview

Superoxide Dismutase (SOD) is a metalloenzyme. It catalyzes the dismutation of superoxide anion to produce hydrogen peroxide (H₂O₂) and oxygen (O₂). As a vital antioxidant enzyme in organisms, SOD is commonly measured via indirect methods combined with various color reactions, because superoxide radicals are unstable and have an extremely short lifespan. Typical chromogenic agents include NBT (Nitro Blue Tetrazolium), WST-1 and WST-8.This product adopts the NBT-based photoreduction method, also known as the NBT photoreduction assay. Its detection principle is as follows: riboflavin undergoes photoreduction in the presence of oxidants. Under aerobic conditions, the reduced riboflavin is rapidly reoxidized to generate superoxide anion radicals (O₂⁻). These radicals reduce Nitro Blue Tetrazolium into blue formazan, which has a strong absorbance at 560 nm. SOD scavenges superoxide anions (O₂⁻) and thus inhibits formazan formation. After the photoreduction reaction, the deeper the blue color of the reaction solution, the lower the SOD activity, and vice versa. The total SOD activity in samples can be calculated through colorimetric analysis using a microplate reader.This method determines SOD activity based on its inhibitory effect on the light-induced reduction of NBT. It is applicable to SOD activity detection in plants, tissues, cells, serum and other specimens. This kit is intended for research use only, and is not suitable for clinical diagnosis or other applications.

Product Components

Catalog No.	Component	50 T	100 T	Storage
T1373360A	SOD Extraction Reagent	250ml	2×250ml	RT
T1373360B	Met Buffer	100ml	2×100ml	2-8 ℃. Store in the dark.
T1373360C	NBT Solution	15ml	30ml	2-8 ℃. Store in the dark.
T1373360D	FD Stock Solution	15ml	30ml	2-8 ℃. Store in the dark.

User-supplied Materials

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, small test tubes, 40 W fluorescent lamp or daylight lamp, illuminance meter, spectrophotometer, cuvettes.

Operating Procedures (For Reference Only)

1. Sample Treatment

① Plasma or Erythrocyte-Containing Samples

Serum or plasma separated from test samples must be free of hemolysis. Remove red blood cells prior to detection if hemolysis occurs. Dilute samples with SOD Extraction Reagent if the readings exceed the detection range.

Simple method for removing red blood cells from serum: Collect blood with anticoagulant tubes and mix by gentle inversion. Take no less than 500 μL whole blood, centrifuge at 3000 rpm for 5 minutes at 4 °C. Transfer the supernatant into a new 1 mL centrifuge tube, dilute with an appropriate amount of normal saline for subsequent testing. Red blood cell lysis solution such as ACK lysis solution can also be used for erythrocyte removal.

② Tissue Samples

Perfuse animal tissues with 0.9% NaCl normal saline containing 20 U/mL Heparin to remove residual blood. Add 500 μL SOD Extraction Reagent per 100 mg tissue. Homogenize the tissue with a glass homogenizer in an ice bath or at 4 °C. Centrifuge the homogenate at 4000 rpm for 10 minutes at 4 °C, and collect the supernatant (crude SOD extract) for enzyme activity assay.

③ Cell Samples

For adherent cells, avoid trypsin digestion to prevent interference with subsequent activity detection. 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, then homogenize with a glass homogenizer in an ice bath or at 4 °C. Centrifuge at 10000 rpm for 10 minutes at 4 °C, and collect the supernatant (crude SOD extract) for detection.

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 under low temperature. Transfer the homogenate into a centrifuge tube. Rinse the mortar or homogenizer with 3 mL SOD Extraction Reagent and combine the rinsing liquid into the same tube, then 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 the enzyme concentration.

⑤ Protein Quantification and Sample Dilution

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

Dilute samples appropriately with the provided SOD Extraction Reagent according to protein concentration and expected dosage. For example, the supernatant of 10% mouse liver tissue homogenate (weight ratio of tissue to homogenate: 10%) usually requires a 10–100 fold dilution.

Store prepared samples in an ice bath if assayed on the same day. For delayed detection, store samples at -20 °C. Assay samples as soon as possible.

2. (Optional) Preparation of SOD Standard

Prepare SOD standard in advance. Dilute the standard with the supplied SOD Extraction Reagent to a series of concentrations: 200, 100, 50, 20, 10, 5, 2 U/mL. Take 20 μL of each diluted standard for testing.

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

3. Light Source Setup

Place the samples under a light incubator or fluorescent lamp. Use an illuminance meter to locate an area with illuminance of 3500–4000 Lx suitable for the photoreaction, and mark the position.

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 ranging from 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 calibrate the absorbance if needed.

6. Sample Loading for SOD Assay

Use a 96-well plate to set up blank control wells, light control wells and test wells following the table below. Add test samples and other reagents sequentially under low light intensity. Mix thoroughly after adding FD Working Solution.

Note: The reaction starts immediately once FD Working Solution is added. Use a multi-channel pipette under low light to minimize errors caused by time differences in reagent addition across wells.

Additive（ul）	Blank Control Tube	Light Control Tube	Test Tube
SOD Extraction Reagent	0.08	0.08	—
Test Sample (Supernatant)	—	—	0.08
NBT Working Solution	2.08	2.08	2.08
FD Working Solution	0.24	0.24	0.24

7. SOD Assay

After mixing thoroughly, place the blank control wells in the dark, and incubate all other wells under 4000 Lx fluorescent light for 20 minutes. Ensure uniform light exposure across all wells. Shorten the reaction time at high temperature and extend it at low temperature. Upon completion of the reaction, set zero using the unilluminated blank control wells immediately, then measure the absorbance at 560 nm with 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. Avoid repeated freeze-thaw cycles for low-temperature stored reagents to prevent inactivation or reduced performance. Test samples can be stored at -70℃for 1 month; repeated freeze-thaw will cause partial loss of SOD activity.

2. Polyphenols in plant samples can trigger irreversible precipitation and deactivation of enzymes. SOD Extraction Reagent contains PVP and other components to effectively remove polyphenols. If the extraction reagent is insufficient, 0.05 M sodium phosphate buffer (pH 7.8) can be used as a substitute.

3. Do not use solutions containing detergents such as Triton X-100 during sample preparation for cells or tissues, as they will interfere with the assay.

4. Antioxidants interfere with the detection. For example, 0.1 mM ascorbic acid and 5 mM GSH will cause a significant increase in measured absorbance.

5. Grind plant samples quickly under ice bath conditions to prevent the decline of SOD activity.

6. When using a spectrophotometer, use cuvettes with a 1 cm optical path. Adjust the volume of supernatant and reagents according to the minimum filling volume of cuvettes.

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

8. Arrange all reaction vessels in a line parallel to the fluorescent tube to ensure consistent light exposure.

9. Maintain the reaction temperature at 25℃, and adjust the illumination time according to SOD activity. Shorten the illumination time at high temperature and prolong it at low temperature.

10. Alternative light sources:

①200 W lamp: Place samples 10–12 cm away and illuminate for 20 min.

②Clean bench (light intensity: 800–1000 Lx at 5 cm): Illuminate for 30–40 min.

③Direct strong sunlight (approx. 30000 Lx): Illuminate for 5–15 min (not recommended).

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

12. Use opened reagents as soon as possible to avoid affecting experimental results.

Storage and Shipping

Storage

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

Shipped In

Wet ice