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超氧化物岐化酶(Superoxide Dismutase,SOD)是含金属辅基的酶，能催化超氧化物阴离子发生岐化作用，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(唑蓝四氮)、WST-1、WST-8等。
本产品是一种基于NBT的光还原反应,所以又称作NBT光还原法，检测原理是在有氧化物质存在下核黄素被光还原,在有氧条件下，被还原的核黄素极易再氧化产生超氧阴离子自由基(O2)，O2可将氮蓝四唑还原为蓝色的甲腙，后者在560nm处有强吸收，而SOD可清除超氧阴离子(O2)，从而抑制了甲腙的形成。于是光还原反应后,反应液蓝色愈深，说明SOD活性愈低，反之酶活性愈高，据此通过酶标仪比色分析就可以计算出样品中总超氧化物岐化酶活性水平。本方法是利用SOD抑制NBT在光照下的还原作用来确定酶活性大小，可用于检测植物、组织、细胞、血清或其它样品中SOD活性。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
产品组分表：
| T1373360 | Component | 50 T | 100 T | Storage |
| T1373360A | SOD 提取试剂 | 250ml | 2×250ml | RT |
| T1373360B | Met缓冲液 | 100ml | 2×100ml | 2-8 ℃. Store in the dark. |
| T1373360C | NBT溶液 | 15ml | 30ml | 2-8 ℃. Store in the dark. |
| T1373360D | FD母液 | 15ml | 30ml | 2-8 ℃. Store in the dark. |
自备材料:
1、生理盐水或PBS、去离子水
2、电子天平、剪刀、低温冰箱或制冰机、冰袋、匀浆器或研钵、离心机、离心管、小试管、40W荧光灯或日光灯、测光仪(照度计)、分光光度计、比色杯
操作步骤（仅供参考）
1、样品处理:
① 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。
② 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提取试剂的比例,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 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 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。
④ 植物样品:准确称取植物材料(果肉或者去叶脉的叶片)0.4g,剪碎,置于4℃预冷的研钵或匀浆器中,加入预冷SOD提取试剂1ml,低温研磨至匀浆后转移至离心管,用3mlSOD提取试剂冲洗研钵或匀浆器并转入离心管,加提取试剂至总体积为4ml,4℃4000r/min 离心20min,上清液为酶提取液,上清液可用于SOD的检测。注意:如果SOD 酶活性较低,应相应减少提取试剂的总体积,以便提高SOD酶的浓度。
⑤ 上述样品准备完毕后可以用BCA法测定蛋白浓度,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(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,用该试剂盒提供的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。
2、(选做)准备SOD标准品:需自备SOD标准品,用本试剂盒提供的SOD提取试剂将SOD标准品稀释至如下系列浓度:200、100、50、20、10、5、2U/ml,各取20μl参考样品进行检测。注意:为避免稀释后SOD酶活性的下降,SOD标准品宜现稀释现使用,本试剂盒对于SOD的检测并不需要SOD作为标准品,但可使用SOD标准品作为阳性对照或作为对SOD活性定量的参考。
3、选取合适的光源:在光照培养箱或日光灯下,用光度仪测定光度值为3500~4000Lx的适合光照反应的位置,做出标记。
4、配制NBT工作液:将Met缓冲液与NBT溶液按23:3的比例混合即可。
5、配制FD工作液:将FD母液和去离子水按2:1~2:3的比例混合,466nm处吸光度为0.30~0.45即可使用,如果不在此范围,可通过增加或减少去离子水的加入量来调节。
6、SOD加样:参考下表使用96孔板设置空白对照孔、光照对照孔、测定孔,在低光强条件下按下表依次加入待测样品和其它各种溶液,加入FD工作液后充分混匀。注意:加入FD溶液后反应即会开始,在低光强条件下用排枪操作可减小各孔间因加入试剂的时间先后差异而导致的误差
加入物(μl) | 空白对照管 | 光照对照管 | 测定管 |
SOD提取试剂 | 0.08 | 0.08 | - |
待测样品(上清液) | - | - | 0.08 |
NBT 工作液 | 2.08 | 2.08 | 2.08 |
FD工作液 | 0.24 | 0.24 | 0.24 |
7、SOD测定:混匀,取空白对照孔置于暗处,其他各孔置于4000Lx日光下反应20min,各孔受光情况应一致,温度高时时间可缩短,低时延长;反应结束后,立即以不照光的空白对照孔调零,用酶标仪测定560nm处吸光度。
计算:
SOD活力单位的定义:以抑制NBT光化还原的50%为一个酶活性单位(U)。
液体中总SOD活力(U/ml)=(A光照-A测定)×V₁/(50%×A光照×VT)
组织、细胞匀浆液中总SOD活力(U/g)=(A光照-A测定)×V/(50%×A光照×VT×W)
血液中总SOD活力(U/gHb)=(A光照-A测定)×V×C/(50%×A光照×VTxHb)
式中:A光照=光照对照孔的吸光度
A测定=测定孔的吸光度
V=样品液总体积(ml)
VT=测定时样品所用体积(ml)
W=样品鲜质量(g)
C=1ml/采血量(ml)
Hb=血红蛋白含量(gHb/ml)
注意事项:
1、上述低温试剂避免反复冻融,以免失效或效率下降。待测样品-70℃可保存1个月,需注意反复冻融会导致SOD部分失活。
2、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。SOD提取试剂含有PVP等成分,可有效去除多酚类物质。SOD提取试剂不够用可以用磷酸钠缓冲液(0.05M,pH7.8)替代。
3、细胞或组织等样品制备时,不能采用含有Triton X-100等去垢剂的溶液,否则会干扰本试剂盒的检测。
4、抗氧化物会对本试剂盒的检测产生干扰,例如0.1mM ascorbic acid,5mM GSH都会使测定出来的吸光度显著升高。
5、对于植物样品,研磨处理应迅速,以免SOD酶活下降,尽量在冰浴条件下处理。
6、如果用分光光度计,比色杯光径应为1cm,加入的上清及试剂量应根据比色杯的最小要求体积而定。
7、所用离心管或96孔板应洁净透明,透光性好。
8、一般要求各孔受光情况一致,所有反应管应排列在与日光灯灯管平行的直线上。
9、反应温度控制在25℃,视酶活性高低适当调整反应时间;温度较高时,光照时间应缩短;温度较低时,光照时间相应延长。
10、如果无4000Lx日光,可200W在10~12cm处,照射20min;超净工作台5cm处光强约800~1000Lx,,照射30~40min;强太阳光下一般光强可达30000Lx,照射5~15min,一般不建议直接用太阳光。
11、为了您的安全和健康,请穿实验服并戴一次性手套操作。
12、试剂开封后请尽快使用,以防影响后续实验效果。
有效期:12个月有效。低温运输,按要求保存。
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
|
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.
|
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)=(Alight control−Atest) )×V₁/ (50%×Alight control×VT)
Total SOD activity in tissue and cell homogenate (U/g)=(Alight control−Atest)×V₁×V/ (50%×Alight control×VT×W)
Parameter explanation
· A<sub>light control</sub>: Absorbance of light control well
· Atest: Absorbance of test well
· V₁: Total volume of reaction solution (mL)
· V: Total volume of sample solution (mL)
· VT: 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.
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危险品化学品经营许可证（带存储）