过氧化物酶（EC1.11.1.7，POD）是生物体内一类含血红素的氧化酶，广泛存在于各种动物、植物和微生物体内，能够催化由过氧化氢参与的多种氧化反应，并且与呼吸作用、光合作用及生长素的氧化等多种生理生化过程密切相关，在细胞代谢的氧化还原过程中起重要作用。
检测原理：过氧化物酶可催化H₂O₂氧化愈创木酚生成茶褐色4-邻甲氧基苯酚，产物在470nm处具有特征吸收峰，通过吸光值变化即可表征过氧化物酶的活性。
适用样本：动植物组织、细胞、细菌、血清（浆）
P1505848
| Component
| 48T | 96T | Storage
|
P1505848A
| Extraction Buffer
| 70 mL
| 70 mL×2
| 2-8℃
|
P1505848B
| Substrate
| 3 mL
| 6 mL
| 2-8℃. Store in the dark.
|
P1505848C
| H₂O₂
| 0.1 mL
| 0.2 mL
| 2-8℃. Store in the dark. |
请在实验前检查各组分的量。
各组分在规格基础上额外提供10%的量用于进行标准曲线制作或预实验。
自备仪器和试剂
自备资源类型
| 资源名称
| 备注
|
仪器
| 酶标仪
| 能测470nm处的吸光度
|
耗材
| 96孔酶标板
| 普通透明板
|
试剂
| PBS（pH7.4）、去离子水
| 标准品及样本稀释用
|
其他
| 匀浆器（组织样本）、恒温箱、制冰机、低温离心机、 可调节式移液枪及枪头
| 检测量较大时合理使用多通道移液器有助于提高实验效率 |
实验流程
1、试剂准备
试剂名称
| 试剂准备
| 注意事项
|
Extraction Buffer
| 即用型；使用前平衡到室温
| 4℃保存
|
Substrate
| 即用型；使用前平衡到室温
| 实验过程中避光放置；4℃避光保存。
|
H₂O₂
| 即用型；使用前平衡到室温
| 实验过程中避光放置；4℃避光保存。 |
2、样本制备
注意：推荐使用新鲜样本，如果不立即进行实验，样本可在-80℃保存1个月。测定过程中样本在冰上放置，以免变性和失活。
2.1 动物组织
用冷的PBS清洗动物组织，吸干组织上的水分，称取约0.1g组织样本，加入1mL Extraction Buffer，冰浴匀浆，8000g，4℃离心10min，取上清液于新离心管中，置冰上待检测。
2.2 植物组织
用冷的PBS清洗植物组织，吸干组织上的水分，尽可能剪碎，称取约0.1g组织样本，加入1mL Extraction Buffer。对于植物纤维不多的植物组织冰浴匀浆，8000g，4℃离心10min，取上清液于新离心管中，置冰上待检测；对于植物纤维较多的植物组织冰浴超声波破碎5min（功率20%或200W，超声3s，间隔7s，重复30次），然后8000g，4℃离心10min，取上清液，置冰上待测。
2.3 细胞或细菌
收集5×10⁶个细胞或细菌，用冷的PBS清洗细胞或细菌，800g离心2min，弃上清，加入1mL Extraction Buffer，冰浴超声波破碎 5min（功率20%或200W，超声3s，间隔7s，重复30次），然后8000g，4℃离心10min，取上清液，置冰上待测。
2.4 血清或血浆
直接检测。
3、实验步骤
3.1 酶标仪准备
预热30min以上，调节波长到470nm。
3.2 工作液的配制
每孔需要190μL工作液，临用前将Extraction Buffer、Substrate、H₂O₂按照每孔139μL：50μL：1μL的比例混匀（按需要检测的样本数量进行计算，可多配一些以防不够）；在37℃（哺乳动物）或25℃（其它物种）预热10min以上；现配现用。
3.3 检测体系配制
在96孔板中加入10μL样本和190μL工作液，混匀。
3.4 吸光度检测
在470nm处读取30s时的吸光值A1和90s时的吸光值A2。
4、结果计算
以下为您提供的推导过程计算公式和简洁计算公式，两者完全相等。
4.1 数据处理
计算ΔA测定=A2-A1。
4.2 样本POD活性的计算
(1) 按样本质量计算
活性单位（U）定义：每g组织在每mL反应体系中每分钟A470变化0.005为一个酶活力单位。 POD (U/g 鲜重)=ΔA测定×V反总÷(W÷V样总×V样)÷0.005÷T=4000×ΔA测定÷W
(2) 按细胞或细菌数量计算
活性单位（U）定义：每10⁴个细胞或细菌在每mL反应体系中每分钟A470变化0.005为一个酶活力单位。
POD(U/10⁴)=ΔA测定×V反总÷(500÷V样总×V样)÷0.005÷T=8×ΔA测定
(3) 按液体体积计算
活性单位（U）定义：每mL液体样本在每mL反应体系中每分钟A470变化0.005为一个酶活力单位。
POD (U/mL)=ΔA测定×V反总÷V样÷0.005÷T=4000×ΔA测定
(4) 按蛋白浓度数量计算
活性单位（U）定义：每mg组织蛋白在每mL反应体系中每分钟A470变化0.005为一个酶活力单位。
POD (U/mg prot)=ΔA测定×V反总÷(Cpr×V样)÷0.005÷T=4000×ΔA测定÷Cpr
参数说明：
V反总：反应体系总体积，0.2mL；
V样：加入样本体积，0.01mL；
V样总：加入提取液体积，1mL；
W：样品质量，g；
T：反应时间，1min；
Cpr：样本蛋白质浓度，mg/mL；
500：细胞或细菌数量，以10⁴为单位。
5、结果展示
示例：取0.121g小鼠肾脏，按照测定步骤操作，用96孔板进行检测。测得ΔA测定=A2-A1=0.159-0.152=0.007。
按样本质量计算得：POD(U/g 鲜重)=ΔA测定×V反总÷(W÷V样总×V样)÷0.005÷T=4000×0.007÷0.121=231.4 U/g 鲜重。
常见问题及解答
Q：检测得到的样本ΔA测定过高或者过低怎么办？
A：如果样本ΔA测定>0.4，则说明样本POD活性太高，需用Extraction Buffer适当稀释，重新进行测定（植物组织建议稀释5倍）。如果样本的ΔA测定＜0.005，可提高样本量或延长反应时间，再重新进行测定。
Q：检测得到的细菌和血清样本过低怎么办？
A：细菌和血清中POD活性较低，建议延长反应时间。
注意事项
1、正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
2、对于组织样本及细胞样本等，可通过测定蛋白浓度来进行样本间结果均一化。推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
3、本试剂盒兼容分光光度计检测，请按照分光光度计检测要求按比例调节检测试剂配制量。
4、生化检测试剂普遍具有刺激性、生物毒性等，为了您的安全和健康，请在实验全程做好生物安全防护， 穿戴实验服、口罩、手套、头套等安全护具，在通风橱、生物安全柜中进行实验。
5、本产品仅供科学研究使用，不适用于临床诊断。
Peroxidase (EC 1.11.1.7, POD) is a class of heme-containing oxidases found widely in animals, plants, and microorganisms. It catalyzes various oxidation reactions involving hydrogen peroxide and is closely related to numerous physiological and biochemical processes such as respiration, photosynthesis, and auxin oxidation, playing a significant role in redox processes during cell metabolism.
Assay Principle
Peroxidase catalyzes the oxidation of guaiacol by H₂O₂, producing a tawny-brown product (tetraguaiacol). This product has a characteristic absorption peak at 470 nm. The change in absorbance is used to determine peroxidase activity.
P1505848
| Component
| 48T | 96T | Storage
|
P1505848A
| Extraction Buffer
| 70 mL
| 70 mL×2
| 2-8℃
|
P1505848B
| Substrate
| 3 mL
| 6 mL
| 2-8℃. Store in the dark.
|
P1505848C
| H₂O₂
| 0.1 mL
| 0.2 mL
| 2-8℃. Store in the dark. |
Please check the volume of all components before the experiment. An extra 10% volume is provided for standard curve preparation or pre-experiments
Required Materials Not Provided
Type
| Name
| Notes
|
Instrument
| Microplate Reader
| Capable of measuring absorbance at 470 nm
|
Consumables
| 96-well microplate
| Standard transparent plate
|
Reagents
| PBS (pH 7.4), Deionized Water
| For diluting standards and samples
|
Others
| Homogenizer (for tissue samples), Incubator, Ice Maker, Refrigerated Centrifuge, Adjustable Pipettes and Tips
| Using a multichannel pipette is recommended for high-throughput assays |
Experimental Procedure
1. Reagent Preparation
Reagent Name
| Preparation
| Notes
|
Extraction Buffer
| Ready-to-use; equilibrate to RT before use
| Store at 4°C
|
Substrate
| Ready-to-use; equilibrate to RT before use
| Protect from light during experiment; store at 4°C protected from light
|
H₂O₂
| Ready-to-use; equilibrate to RT before use
| Protect from light during experiment; store at 4°C protected from light |
2. Sample Preparation
Note: Fresh samples are recommended. If not used immediately, samples can be stored at -80°C for up to 1 month. Keep samples on ice during assay preparation to prevent denaturation and inactivation.
2.1 Animal Tissue
Rinse tissue with ice-cold PBS, blot dry.
Weigh about 0.1 g of tissue, add 1 mL of Extraction Buffer.
Homogenize in an ice bath.
Centrifuge at 8,000 g, 4°C for 10 min.
Collect the supernatant into a new tube and keep on ice for assay.
2.2 Plant Tissue
Rinse tissue with ice-cold PBS, blot dry, and mince thoroughly.
Weigh about 0.1 g of tissue, add 1 mL of Extraction Buffer.
For tissues with low fiber content: Homogenize in an ice bath, then centrifuge as in 2.1.
For fibrous tissues: Sonicate in an ice bath for 5 min (20% power or 200 W, 3s pulse, 7s interval, 30 cycles), then centrifuge as in 2.1.
Collect supernatant and keep on ice.
2.3 Cells or Bacteria
Collect 5×10⁶ cells/bacteria.
Wash with ice-cold PBS, centrifuge at 800 g for 2 min, discard supernatant.
Add 1 mL Extraction Buffer.
Sonicate in an ice bath (parameters as in 2.2 fibrous tissues).
Centrifuge at 8,000 g, 4°C for 10 min.
Collect supernatant and keep on ice.
2.4 Serum or Plasma
3. Assay Procedure
3.1 Microplate Reader Preparation
3.2 Working Solution Preparation
Prepare the Working Solution fresh before use.
For each well, mix 139 µL Extraction Buffer, 50 µL Substrate, and 1 µL H₂O₂ (total 190 µL per well).
Prepare a master mix based on the number of samples (+ extra volume).
Pre-warm the Working Solution at 37°C (mammalian) or 25°C (other species) for at least 10 min.
3.3 Assay Setup
3.4 Absorbance Measurement
4. Calculation of Results
The following derived and simplified formulas are equivalent.
4.1 Data Processing
4.2 Sample POD Activity Calculation
(1) Based on Sample Weight:
Activity Unit (U) Definition: One unit of enzyme activity is defined as the amount that causes a change of 0.005 in A470 per minute per gram of tissue per mL of reaction mixture.
Derivation Formula: POD (U/g fresh weight) = ΔA × Vtotal reaction ÷ (W ÷ Vtotal extract × Vsample) ÷ 0.005 ÷ T
Simplified Formula: POD (U/g fresh weight) = 4000 × ΔA / W
(2) Based on Cell/Bacteria Count:
Activity Unit (U) Definition: One unit of enzyme activity is defined as the amount that causes a change of 0.005 in A470 per minute per 10<sup>4</sup> cells or bacteria per mL of reaction mixture.
Derivation Formula: POD (U/10<sup>4</sup>) = ΔA × Vtotal reaction ÷ (500 ÷ Vtotal extract × Vsample) ÷ 0.005 ÷ T
Simplified Formula: POD (U/10<sup>4</sup>) = 8 × ΔA
(3) Based on Liquid Volume:
Activity Unit (U) Definition: One unit of enzyme activity is defined as the amount that causes a change of 0.005 in A470 per minute per mL of liquid sample per mL of reaction mixture.
Derivation Formula: POD (U/mL) = ΔA × Vtotal reaction ÷ Vsample ÷ 0.005 ÷ T
Simplified Formula: POD (U/mL) = 4000 × ΔA
(4) Based on Protein Concentration:
Activity Unit (U) Definition: One unit of enzyme activity is defined as the amount that causes a change of 0.005 in A470 per minute per mg of tissue protein per mL of reaction mixture.
Derivation Formula: POD (U/mg prot) = ΔA × Vtotal reaction ÷ (Cpr × Vsample) ÷ 0.005 ÷ T
Simplified Formula: POD (U/mg prot) = 4000 × ΔA / Cpr
Parameter Definitions:
ΔA: Change in absorbance (A₂ - A₁)
Vtotal reaction: Total reaction volume, 0.2 mL
Vsample: Volume of sample added to well, 0.01 mL
Vtotal extract: Total volume of extraction buffer added, 1 mL
W: Sample weight, g
T: Reaction time, 1 min
Cpr: Sample protein concentration, mg/mL
500: Cell/Bacteria number, in units of 10⁴ (e.g., 5×10⁶ cells = 500 × 10⁴)
5. Results Example
Sample: 0.121 g mouse kidney.
Measured: A₁ = 0.152, A₂ = 0.159.
ΔA = 0.159 - 0.152 = 0.007.
POD (U/g fresh weight) = 4000 × 0.007 / 0.121 = 231.4 U/g fresh weight
Frequently Asked Questions (FAQ)
Q: What should I do if the sample ΔA is too high or too low?
A: If ΔA > 0.4, the POD activity is too high. Dilute the sample appropriately with Extraction Buffer and re-assay (for plant tissues, a 5-fold dilution is recommended). If ΔA < 0.005, increase the sample amount or extend the reaction time, then re-assay.
Q: What if the ΔA for bacterial and serum samples is too low?
A: POD activity in bacteria and serum is typically low. It is recommended to extend the reaction time.
Precautions
1. It is recommended to perform a pilot experiment with 2-3 samples expected to have significant differences before formal testing.
2. For tissue samples, cell samples, and others, results can be normalized between samples by determining the protein concentration. We recommend using the Aladdin B665595 BCA Protein Quantification Kit or the R1491648 Ready-to-Use BCA Protein Quantification Kit.
3. This kit is compatible with spectrophotometer detection. Adjust the preparation volume of detection reagents proportionally according to the spectrophotometer's requirements.
4. Biochemical reagents are generally irritating and/or toxic. For your safety and health, please wear appropriate personal protective equipment (lab coat, mask, gloves, head cover, etc.) throughout the experiment and perform steps in a fume hood or biosafety cabinet when applicable.
5. This product is for research use only. Not for use in diagnostic procedures.
ZJ26F0737383
危险品化学品经营许可证（带存储）