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超氧化物歧化酶 (SOD)提取试剂
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
超氧化物歧化酶 (Superoxide Dismutase, SOD)是含金属辅基的酶，能催化超氧化物阴离子发生歧化作用，生成过氧化氢(H₂O₂)和氧气 (O₂)，是生物体内一种重要的抗氧化酶，由于超氧自由基是不稳定的的自由基，寿命极短，SOD活性一般用间接方法测定，并利用各种呈色反应来测定SOD活力，其中显色剂有NBT (四氮唑蓝)、WST-1、WST-8等。
超氧化物歧化酶 (SOD)提取试剂主要有磷酸盐、酚类物质去除剂等组成，用于裂解动植物组织样本、细胞样本，提取样品中的超氧化物歧化酶。植物中的多酚类物质会引起酶蛋白不可逆沉淀，使酶失去活性，因此在提取SOD酶时，必须添加多酚类物质的吸附剂，将多酚物质除去，避免酶蛋白变性失活。本产品可有效避免此类情况。该试剂仅用于科研领域，不适用于临床诊断或其他用途。
自备材料：
1、蒸馏水
2、离心管或试管、匀浆器或研钵、低温离心机
操作步骤 (仅供参考)：
1、植物组织样品：取正常或逆境下的新鲜植物组织，清洗干净，擦干，切碎，迅速称取，按植物组织：超氧化物歧化酶 (SOD)提取试剂=0.5g: 2ml的比例，加入预冷的超氧化物歧化酶 (SOD)提取试剂，冰浴情况下充分捣碎或研磨，用超氧化物歧化酶 (SOD)提取试剂冲洗研钵或匀浆器，合并冲洗液至该离心管，补加超氧化物歧化酶 (SOD)提取试剂至10ml，4℃ 4000r/min离心10min，取上清液 (SOD粗提液)用于酶活性的测定。
2、动物组织样品：动物用含有20U/ml Heparin的生理盐水 (0.9% NaCl containing 20U/ml Heparin)灌流清除血液后获取组织样品；按照每100mg组织加入500μl SOD检测缓冲液的比例，用玻璃匀浆器在4℃或冰浴匀浆，4℃ 4000r/min离心10min，取上清液 (SOD粗提液)用于酶活性的测定。
3、血浆或含红细胞的样品：从待测样品中分理出的血清或血浆不应有溶血，如果含有应去除红细胞后检测，如超过检测范围，用超氧化物歧化酶 (SOD)提取试剂稀释后检测；血清去除红细胞的简易方法如下：用抗凝管收集血液，颠倒混匀，取至少500μl全血，4℃ 3000r/min离心5min，转移上清至另一新的1ml离心管中，适量生理盐水稀释后待测，亦可采用红细胞裂解液去除红细胞，如ACK红细胞裂解液等。
4、细胞样品：对于贴壁细胞，由于后续用于酶活性的测定，避免使用胰酶消化细胞。可以使用细胞刮或EDTA处理细胞并收集细胞，细胞用无菌的PBS或生理盐水洗涤1次，按照每106细胞加入300-500μl超氧化物歧化酶 (SOD)提取试剂的比例，用玻璃匀浆器在4℃或冰浴匀浆，4℃ 10000r/min离心10min，取上清液 (SOD粗提液)用于酶活性的测定。
5、上述样品准备完毕后可以BCA蛋白定量检测试剂盒测定蛋白浓度，通常10-20μg蛋白的细胞或组织匀浆液样品其中的SOD平均活力约1个活力单位左右 (不同细胞和组织的差异会比较大，该活力范围仅作为初步的参考)；每种样品准备20-100μg蛋白量通常已经足够用于后续检测；根据蛋白浓度和预计的蛋白使用量，用超氧化物歧化酶 (SOD)提取试剂适当稀释样品。例如小鼠肝脏组织10%匀浆液 (组织和匀浆液的重量比为10%)上清，通常需要稀释10-100倍，准备好的样品如果当天测定，可以冰浴保存；如果当天不能完成测定，可以-20℃冻存，但建议尽量当天完成测定。
6、如果需要测定SOD活性，可参考相关产品说明书。
计算：
样品粗酶液获得率 (ml/g)=上清液体积 (ml)/样品质量 (g)×100%
注意事项：
1、待测样品中不能含有磷酸酶抑制剂，同时需避免反复冻融。
2、所测样本的值高于标准曲线的上限，用提取试剂稀释样品后重新测定。
3、为了您的安全和健康，请穿实验服并戴一次性手套操作。
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.
规格
别名
超氧化物歧化酶提取试剂 | SOD提取试剂
英文别名
Superoxide Dismutase Extraction Reagent | SOD Extraction Reagent
规格或纯度
BioReagent, 哺乳动物样品抽提用, 植物样品抽提用
稳定性与储存
Store at room temperature long term (12 months).
英文名称
Superoxide Dismutase (SOD) Extraction Reagent
储存条件
室温
运输条件
常规运输
产品线
生物产品
名称和识别符
分子类型
生物试剂/缓冲液
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高级数据
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0332671 |
分析证书 | S1509588 |
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