Superoxide Dismutase (SOD) is a metalloenzyme that catalyzes the dismutation of superoxide anions to produce hydrogen peroxide (H₂O₂) and oxygen (O₂). It serves as a crucial antioxidant enzyme in living organisms. Given that superoxide free radicals are unstable and have an extremely short half-life, SOD activity is generally determined by indirect methods, which rely on various chromogenic reactions. Common chromogenic agents include Nitrotetrazolium Blue (NBT), WST-1, and WST-8.
The Superoxide Dismutase (SOD) Extraction Reagent is mainly composed of phosphates, polyphenol removers, and other components. It is used for lysing animal and plant tissue samples as well as cell samples to extract SOD from the specimens. Polyphenols in plants can induce irreversible precipitation of enzyme proteins, leading to enzyme inactivation. Therefore, during the extraction of SOD, it is essential to add polyphenol adsorbents to eliminate polyphenols and prevent the denaturation and inactivation of the enzyme protein. This product can effectively avoid such issues. Note: This reagent is intended for research purposes only and not for clinical diagnosis or any other applications.
Materials to Be Prepared by the User
1. Distilled water
2. Centrifuge tubes or test tubes, homogenizers or mortars, low-temperature centrifuges
Operating Procedures (For Reference Only)
1. Plant Tissue Samples
Take fresh plant tissues under normal or stress conditions, rinse them thoroughly, dry them, and cut them into small pieces. Weigh the tissues quickly and mix them with pre-cooled SOD Extraction Reagent at a ratio of 0.5 g tissue to 2 ml reagent. Homogenize or grind the mixture sufficiently on ice bath. Rinse the mortar or homogenizer with the SOD Extraction Reagent and transfer the rinsing solution into the centrifuge tube. Add additional SOD Extraction Reagent to bring the total volume to 10 ml. Centrifuge the mixture at 4000 r/min for 10 min at 4℃. Collect the supernatant (crude SOD extract) for enzyme activity assay.
2. Animal Tissue Samples
Perfuse the animals with normal saline containing 20 U/ml Heparin (0.9% NaCl containing 20 U/ml Heparin) to remove blood, then harvest the tissue samples. Add 500 μl of SOD Assay Buffer per 100 mg of tissue and homogenize the mixture using a glass homogenizer at 4℃ or on ice bath. Centrifuge the homogenate at 4000 r/min for 10 min at 4℃. Collect the supernatant (crude SOD extract) for enzyme activity assay.
3. Plasma or Erythrocyte-containing Samples
The serum or plasma isolated from the test samples should be free of hemolysis; if hemolysis occurs, remove the erythrocytes before the assay. If the SOD activity exceeds the detection range, dilute the samples with the SOD Extraction Reagent before testing. A simple method for removing erythrocytes from serum is as follows: Collect blood in an anticoagulant tube and invert the tube gently to mix well. Take at least 500 μl of whole blood and centrifuge it at 3000 r/min for 5 min at 4℃. Transfer the supernatant into a new 1 ml centrifuge tube, dilute it with an appropriate volume of normal saline, and proceed with the assay. Alternatively, erythrocyte lysis buffers (e.g., ACK Lysis Buffer) can be used to eliminate erythrocytes.
4. Cell Samples
For adherent cells, avoid using trypsin for cell detachment since the cells will be used for subsequent enzyme activity assay. Instead, harvest the cells using a cell scraper or EDTA treatment. Wash the collected cells once with sterile PBS or normal saline. Add 300–500 μl of SOD Extraction Reagent per 10⁶ cells and homogenize the suspension using a glass homogenizer at 4℃ or on ice bath. Centrifuge the homogenate at 10000 r/min for 10 min at 4℃. Collect the supernatant (crude SOD extract) for enzyme activity assay.
5. After preparing the above samples, determine the protein concentration using a BCA Protein Quantification Kit. Generally, the average SOD activity in 10-20 μg of protein from cell or tissue homogenates is approximately 1 activity unit (Note: Significant variations exist among different cell and tissue types; this activity range is provided for preliminary reference only). Preparing 20-100 μg of protein per sample is usually sufficient for subsequent assays. Dilute the samples appropriately with the SOD Extraction Reagent according to the measured protein concentration and the intended protein dosage for the assay. For example, the supernatant of a 10% homogenate of mouse liver tissue (weight ratio of tissue to homogenate is 10%) typically requires 10-100-fold dilution. Prepared samples can be stored on ice bath for the assay on the same day; if the assay cannot be completed on the same day, the samples can be stored at -20℃, but it is recommended to perform the assay as soon as possible.
6. For the determination of SOD activity, refer to the instruction manual of the corresponding kit.
Calculation
Yield rate of crude enzyme solution (ml/g) = Volume of supernatant (ml) / Weight of sample (g) × 100%
Precautions
1. The test samples must not contain phosphatase inhibitors, and repeated freeze-thaw cycles of the samples should be avoided.
2. If the measured value of a sample exceeds the upper limit of the standard curve, dilute the sample with the extraction reagent and perform the assay again.
3. For your safety and health, wear a lab coat and disposable gloves during operation.
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