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HRP标记试剂（高碘酸钠法）盒采用简便快捷的流程，实现抗体的HRP标记。该试剂盒提供标记100 µg和1 mg抗体所需的所有试剂，以及详细的分步操作指南。
本试剂盒提供的Activated HRP具有胺反应性，利用高碘酸钠氧化法活化辣根过氧化物酶（Horseradish Peroxidase，HRP）的糖基，经过活化的HRP带有醛基可自发有效地与抗体或其他蛋白质上的伯胺交联，发生希夫碱反应（Schiff's base reaction）。本试剂盒适用于带有游离氨基的物质的标记，比如：带有游离氨基（伯胺）的抗体、蛋白或者小分子化合物，可简单、快速地实现抗体与 HRP 的偶联，偶联试剂盒产品稳定性好，随用随取，方便快捷。
使用方法
1. 抗体准备
1.1 抗体推荐使用浓度为2-5 mg/mL，如果抗体浓度低于2 mg/ml，标记效率可能会降低。待标记的抗体或蛋白最好溶解于无氨的缓冲液中（如：MES，MOPS，HEPES，PBS），pH 为 6.5-8.5，且溶液中无叠氮钠等防腐剂（叠氮钠会抑制辣根过氧化物酶（HRP）活性，可能干扰后续应用），若抗体中含有这些物质，推荐使用透析或者超滤换液方法将抗体换液至0.01M PBS（pH 7.0-7.4）中。
2. 缓冲液配制
2.1 1 M 碳酸钠-碳酸氢钠缓冲液的配制：向装有碳酸钠-碳酸氢钠粉末的试管中加入 1 mL 超纯水。盖紧管盖，轻轻摇晃直至粉末完全溶解，即可获得 1 M 碳酸钠-碳酸氢钠缓冲液（pH 9.2）。用超纯水将1 M溶液稀释10倍，制备0.1 M 碳酸钠-碳酸氢钠缓冲液。注意： 配制好的缓冲液在 2-8°C 条件下储存，可保存1-2周。
3. 抗体与HRP偶联步骤
以下实验操作以P1508744规格示例，其他规格可以按照倍数增加进行调整。
3.1 配制HRP溶液（5 mg/mL）：向Activated HRP中加入20 μL超纯水，轻微涡旋直至全部溶解。注：复溶的Activated HRP溶液须现用现配；
3.2 将100 µg 准备好的抗体加入配制的HRP溶液中，向反应液中加入7 µL体积的1 M 碳酸钠-碳酸氢钠缓冲液，充分混合，室温避光反应2h；
3.3 配制氰基硼氢化钠溶液：向氰基硼氢化钠中加入400 µL 0.1 M 碳酸钠-碳酸氢钠缓冲液，盖紧管盖，轻轻摇晃直至粉末完全溶解。注：氰基硼氢化钠溶液须现用现配；
3.4 向3.2混合液中加入7 µL 氰基硼氢化钠溶液，4℃避光反应30 min后，即可得到偶联抗体。可供选择将 HRP-抗体偶联物转移至收集管中保存。
偶联物保存
向HRP-抗体偶联物中添加50%甘油，-20℃避光保存。
注意事项
1. 收到本产品后请立即使用或按照说明书推荐的条件保存。随着保存时间的延长，Activated HRP的酶活力和标记能力可能会有一 定程度的下降；
2. 有关抗体的具体用量需要根据具体要求通过优化实验来决定，一般情况下，HRP:抗体比例越高，抗体标记的效率越高，抗体上标记的 HRP 越多，灵敏度，信噪比会越好，但是标记太多也可能屏蔽抗体的活性位点，故 HRP:抗体的质量比可以在 0.5:1~2:1 之间调试。我们推荐使用HRP:抗体的质量比为1:1；
3. 在正式实验前，需将试剂恢复至室温，较低的温度会影响偶联效果；
4. 操作过程中，务必穿戴实验服、一次性手套及防护装备；
5. 所有产品仅限于研究用途。
Introduction
HRP Conjugation Kit (Periodate Activated) uses a simple and quick process for HRP conjugation of antibodies. The kit provides all the reagents needed to label to 100 µg and 1 mg of antibody, as well as a detailed step-by-step protocol.
This kit provides activated HRP with amine reactivity. The horseradish peroxidase (HRP) is activated via the sodium periodate oxidation method, which targets the glycosyl groups on HRP. The resulting activated HRP carries aldehyde groups that can spontaneously and efficiently cross‑link with primary amines on antibodies or other proteins through a Schiff’s base reaction. The kit is suitable for labeling substances containing free amino groups, such as antibodies, proteins, or small‑molecule compounds bearing primary amines. It enables simple and rapid conjugation of antibodies with HRP. The kit offers excellent stability, is ready to use on demand, and ensures a convenient and efficient workflow.
Instruction for use
1.1 The recommended antibody concentration for labeling is 2–5 mg/mL. Labeling efficiency may be reduced if the antibody concentration is below 2 mg/mL. The antibody or protein to be labeled should be dissolved in an amine‑free buffer (e.g., MES, MOPS, HEPES, or PBS) at a pH of 6.5–8.5. The solution must be free of preservatives such as sodium azide, which can inhibit horseradish peroxidase (HRP) activity and interfere with downstream applications. If the antibody solution contains such interfering substances, it is recommended to exchange the buffer to 0.01 M PBS (pH 7.0–7.4) using dialysis or ultrafiltration prior to labeling.
2. Prepare the reagents
2.1 Prepare a 1 M Sodium Carbonate-Bicarbonate buffer: Add 1 mL of ultrapure water to the Sodium Carbonate–Bicarbonate powder. Cap the tube and shake until the powder is completely dissolved to obtain 1 M Sodium Carbonate–Bicarbonate buffer (pH 9.2). Prepare 0.1 M Sodium Carbonate-Bicarbonate buffer by diluting the 1 M solution 10-fold with ultrapure water. Note: The prepared Buffer is stable for 1-2 weeks when stored at 2-8°C.
3. Antibody-HRP Conjugation Procedure
The following experimental procedure is illustrated for the P1508744 specification. For other specifications, scale the reaction proportionally based on the provided guidelines.
3.1 Prepare HRP solution (5 mg/mL)—Add 20 μL of Ultrapure water to Activated HRP. Vortex gently until the solution is homogenous. Note: The Reconstituted SMCC Activated HRP solution must be prepared fresh before each use.
3.2 Add 100 µg of the prepared antibody to the prepared HRP solution. Add 7 µL of 1 M Sodium Carbonate-Bicarbonate buffer to the reaction mixture. Mix thoroughly and incubate at room temperature, protected from light, for 2 hours.
3.3 Preparation of Sodium Cyanoborohydride solution, Add 400 µL of 0.1 M Sodium Carbonate-Bicarbonate buffer to the Sodium Cyanoborohydride tube. Cap the tube tightly and gently vortex or invert until the powder is completely dissolved. Note: The Sodium Cyanoborohydride solution must be prepared fresh before each use.
3.4 In a fume hood, add 7 µL of Sodium Cyanoborohydride solution to the mixture in step 3.2. Incubate the reaction at 4°C in the dark for 30 minutes to obtain the conjugated antibody. For storage, the conjugate can be transferred to the Collection Tube.
Storage
Add 50% glycerol to the HRP-antibody conjugate and store at -20°C protected from light.
Matters needing attention
1. Upon receipt of this product, it should be used immediately or stored according to the recommended conditions. With prolonged storage, the enzymatic activity and labeling capability of the Activated HRP may decrease to some extent.
2. The optimal antibody quantity should be determined experimentally based on specific requirements. Generally, a higher HRP-to-antibody ratio increases labeling efficiency and the number of HRP molecules conjugated per antibody, leading to improved sensitivity and signal-to-noise ratio. However, excessive labeling may block the active sites of the antibody and reduce its immunoreactivity. Therefore, the HRP-to-antibody mass ratio can be optimized within the range of 0.5:1 to 2:1. We recommend starting with a mass ratio of 1:1 for most applications.
3. Before formal experimentation, allow all reagents to reach room temperature, as lower temperatures may impair conjugation efficiency.
4. During operation, always wear a lab coat, disposable gloves, and protective equipment.
5. All products are for research use only.
| P1506712 | Component | 1 reaction | Storage | Quantity Per reaction |
| P1506712A | Activated HRP | 1 mg | -20℃. Store in the dark. | 1 mg for labeling 1 mg of antibody |
| P1506712B | 碳酸钠-碳酸氢钠 | 1 vial | RT. | Prepare according to instructions |
| P1506712C | 氰基硼氢化钠 | 1 vial | RT. | Prepare according to instructions |
| P1506712D | 收集管 | 1 EA | RT. | 1 EA for 1 reaction |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0332799 |
分析证书 | P1506712 |
| ZJ26F0332800 |
分析证书 | P1506712 |
| ZJ26F0332801 |
分析证书 | P1506712 |
| ZJ26F0332802 |
分析证书 | P1506712 |
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