辣根过氧化物酶是一种具有氧化还原酶属性的血红蛋白，是临床化学试剂中的常用酶，可与抗体、抗原或链霉亲和素等进行偶联，广泛应用于临床诊断、免疫检测、化学发光、ELISA、免疫组化及生化研究等。
一、产品基本信息
1. 名称：辣根过氧化物酶（Horseradish Peroxidase，HRP）
2. 来源：植物辣根（Armoracia rusticana）根部提取
3. 分子量：约 40 kDa（单链多肽，含 4 个二硫键）
4. 活性单位：≥300 单位 / 毫克
5. 纯度指标：RZ 值（Reinheit Zahl）≥3.0（反映血红蛋白与酶蛋白的纯度比）
6. 储存条件：-20℃干燥避光保存，复溶后 4℃稳定 6 周，长期需分装 -70℃冻存。
二、理化性质
1. 外观：棕褐色结晶物质或冻干粉
2. 溶解性：易溶于水，在 58% 硫酸铵饱和度以下可溶
3. 稳定性：
• pH 范围：3.0-9.0（等电点）
• 耐温性：63℃加热 15 分钟仍保持活性
4. 抑制剂：叠氮化钠、氰化物、重金属离子（Cd²+、Cu²+ 等）
三、功能与应用
1. 催化机制HRP 催化过氧化氢（H₂O₂）参与的氧化反应，将底物（如 DAB、TMB 等）转化为显色产物。
2. 主要应用领域
| 应用类型 | 具体用途 | 示例方法 |
|---|
| 检测免疫 | ELISA、Western Blot、免疫组化（IHC）的抗体标记 | HRP 标记二抗 + 链亲和素 |
| 分子生物学 | 核酸杂交（Southern/Northern Blot）、EMSA 的探针标记 | 生物素 - 链霉亲和素系统 |
| 生化分析 | 葡萄糖过氧化物定量检测 | 比色法 / 化学发光法 |
| 病理研究 | 神经元追踪（轴浆运输示踪） | HRP 逆行标记法 |
四、使用指南
1. 标记操作（以抗体标记为例）
• 标记方法：戊二醛交联法、过碘酸氧化法
• 推荐比例：HRP: 抗体 = 1:1-1:3（摩尔比）
• 纯化步骤：凝胶过滤层析去除游离酶
2. 显色体系
| 底物 | 显色产物 | 检测方式 | 适用场景 |
|---|
| DAB | 棕色沉淀 | 光学显微镜 / 化学成像 | 免疫组化、Western Blot |
| TMB | 蓝色可溶物（酸化后变黄） | 酶标仪（450 nm） | ELISA |
| ECL 化学发光剂 | 发光信号 | X 光片 / 化学发光成像仪 | 高灵敏度 Western Blot |
五、注意事项
1. 避免反复冻融：分装后 - 70℃保存，活性避免损失；
2. 抑制剂干扰：避免使用含叠氮钠（NaN₃）的缓冲液；
3. 安全防护：接触 DAB 时需佩戴手套，废液按有害化学物质处理；
4. 优化建议：不同应用需调整 HRP 浓度（如 Western 推荐 1:2000-1:10000 稀释）。
Horseradish Peroxidase (HRP) is a hemoglobin with redox properties, a common reagent in clinical chemistry. It can conjugate with antibodies, antigens, or streptavidin, and is widely used in clinical diagnosis, immunoassays, chemiluminescence, ELISA, immunohistochemistry, and biochemical research.
I. Basic Product Information
1. Name: Horseradish Peroxidase (HRP)
2. Source: Extracted from the roots of Armoracia rusticana (horseradish)
3. Molecular weight: Approximately 40 kDa (single-chain polypeptide, containing 4 disulfide bonds)
4. Activity unit: ≥300 units/mg
5. Purity index: RZ value (Reinheit Zahl) ≥3.0 (reflects the purity ratio of hemoglobin to enzyme protein)
6. Storage conditions: Store at 20°C in a dry, dark place. After reconstitution, it remains stable for 6 weeks at 4°C; for long-term storage, aliquot and keep at -70°C.
II. Physicochemical Properties
1. Appearance: Brown crystalline substance or lyophilized powder
2. Solubility: Soluble in water; soluble below 58% ammonium sulfate saturation
3. Stability:
• pH range: 3.0-9.0 (isoelectric point)
• Thermostability: Retains activity after heating at 63°C for 15 minutes
4. Inhibitors: Sodium azide, cyanide, heavy metal ions (e.g., Cd²+, Cu²+)
III. Functions and Applications
1. Catalytic mechanismHRP catalyzes oxidation reactions involving hydrogen peroxide (H₂O₂), converting substrates (e.g., DAB, TMB) into chromogenic products.
2. Main application fields
| Application Type | Specific Uses | Example Methods |
|---|
| Detection Immunology | Antibody labeling for ELISA, Western Blot, Immunohistochemistry (IHC) | HRP-labeled secondary antibody + streptavidin |
| Molecular Biology | Probe labeling for nucleic acid hybridization (Southern/Northern Blot), EMSA | Biotin-streptavidin system |
| Biochemical Analysis | Quantitative detection of glucose peroxide | Colorimetry/chemiluminescence |
| Pathological Research | Neuron tracing (axoplasmic transport tracing) | HRP retrograde labeling |
IV. Usage Guidelines
1. Labeling operation (taking antibody labeling as an example)
• Labeling methods: Glutaraldehyde cross-linking, periodate oxidation
• Recommended ratio: HRP:antibody = 1:1-1:3 (molar ratio)
• Purification step: Remove free enzyme via gel filtration chromatography
2. Chromogenic systems
| Substrate | Chromogenic Product | Detection Method | Application Scenario |
|---|
| DAB | Brown precipitate | Light microscope/chemical imaging | Immunohistochemistry, Western Blot |
| TMB | Blue soluble substance (turns yellow after acidification) | Microplate reader (450 nm) | ELISA |
| ECL chemiluminescent reagent | Luminescent signal | X-ray film/chemiluminescence imager | High-sensitivity Western Blot |
V. Precautions
1. Avoid repeated freeze-thaw cycles: Aliquot and store at -70°C to prevent activity loss
2. Inhibitor interference: Avoid using buffers containing sodium azide (NaN₃)
3. Safety protection: Wear gloves when handling DAB; dispose of waste liquid as hazardous chemical waste
4. Optimization suggestion: Adjust HRP concentration according to different applications (e.g., 1:2000-1:10000 dilution is recommended for Western Blot)
ZJ26F0838479
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