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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。
HRP标记试剂盒（SMCC法）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抗体所需的所有试剂和纯化柱，以及详细的分步操作指南。
HRP标记试剂盒（SMCC法）是一种含巯基反应性标记试剂盒。试剂盒提供共轭试剂，该试剂与伯胺(-NH₂)反应，为抗体引入巯基基团(-SH)，之后，抗体上的巯基可与修饰后的HRP形成共价键，从而快速实现HRP与抗体的偶联。
图1 HRP标记试剂盒（SMCC法）（M1508746）标记原理图
使用方法
1. 抗体处理
向抗体添加巯基：使用共轭试剂，该试剂可修饰伯胺。确保抗体具有游离的巯基， 以便与 SMCC Activated HRP的马来酰亚胺基团发生反应。
1.1 向抗体中加入Conjugation Buffer使抗体浓度调整至3-5 mg/mL。建议采用较高蛋白浓度，若浓度低于3 mg/mL，则需对抗体进行浓缩处理；若IgG非无盐状态或已处于溶液中，需通过浓缩换液或者透析法将抗体置换至 Conjugation Buffer；
1.2 向Conjugation Reagent中加入1mL 的纯水，混匀；注：Conjugation Reagent溶液须现用现配；
1.3 每100 μg抗体溶液中加入1 µL Conjugation Reagent储备液，涡旋混合。室温孵育反应30 min；
1.4 在孵育期间准备脱盐柱；
1.4.1 打开脱盐柱底部，将脱盐柱放入配套的离心管中，打开脱盐柱盖，1000 x g离心2 min，弃去洗脱液；
注意：使用非水平转头的情况下，由于离心会使树脂压实形成一个向上的斜面，该斜面的方向宜在后续步骤中保持，所以在脱盐柱外壳上的斜面向上位置做标记，在随后的离心步骤中需要调整好离心管的放入方向，确保离心后斜面的方向和位置不会改变。
1.4.2 加入500 µL Conjugation Buffer，1000 x g离心2 min，弃去洗脱液；
1.4.3 重复步骤1.4.2两次，第三次洗涤时加入Conjugation Buffer 直到进入下一步骤时再进行离心；
1.4.4 第三次洗涤离心后，脱盐柱中应无溶液残留，如有残留，可短暂离心直至溶液完全排出；
1.5 将准备好的脱盐柱放入收集管中，并将步骤1.3中的全部样品加入脱盐柱中，若体积不足60 µL，用Conjugation Buffer补足；
1.6 1000 × g离心2 min，保留洗脱液，得到巯基化的IgG；
2. 与HRP进行偶联
2.1 向0.1 mg SMCC Activated HRP粉末中加入1.6中巯基化的抗体，充分混合，室温避光孵育1 h；
2.2 阻断过量的游离巯基
2.2.1 向Quencher Reagent中加入500 µL DMSO，涡旋混匀；
2.2.2 向步骤2.2制备的偶联混合液中加入1 µL Quencher reagent ，充分混合，室温避光孵育20 min；
2.3· 偶联反应已完成。反应混合物中应主要含有HRP-抗体偶联物，并含有少量游离的HRP和抗体。未偶联的HRP不应干扰检测。
偶联物保存
向HRP-抗体偶联物添加50%甘油，-20℃避光保存。
注意事项
1. 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 NaN3 等防腐剂（若含有此类物质需先纯化后，再标记）；
2. 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；
3. 脱盐柱建议一次性使用；
4. 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；
5. 操作过程中，务必穿戴实验服、一次性手套及防护装备；
6. 所有产品仅限于研究用途。
HRP Conjugation Kit (SMCC Activated) uses a simple and quick process for HRP conjugation of antibodies. The kit provides all the reagents, purification columns needed to label to 100 μg of antibody, as well as a detailed step-by-step protocol.
The HRP Conjugation Kit (SMCC Method) is a SH-reactive labeling kit. The kit provides a Conjugation Reagent that reacts with primary amines (-NH₂) to introduce a SH group onto the antibody. Subsequently, Modified HRP and sulfhydryl group on the antibody form a covalent bond during conjugation.
Fig. 1 HRP Conjugation Kit (SMCC Method) (M1508746) Labeling Principle Schematic
Instruction for use
1. Antibody sulfhydryl modification
To add sulfhydryls to antibodies: Use Conjugation Reagent, which modify primary amines. Ensure antibodies have a free sulfhydryl to react with the maleimide group of SMCC Activated HRP.
1.1 Add Conjugation Buffer to dissolve the antibody to achieve a concentration of 3–5 mg/mL. A higher protein concentration is preferred. If less than 3 mg/mL, the antibody should be concentrated. If the IgG is not salt-free or if it is already in solution, the antibody must be transferred to the Conjugation Buffer via concentration exchange or dialysis.
1.2 Add 1 mL of purified water to the Conjugation Reagent and mix thoroughly. Note: The Conjugation Reagent solution must be prepared fresh before each use.
1.3 Add 1 µL of prepared Conjugation Reagent per 100 μg of antibody solution, briefly vortex to mix.
1.4 During the incubation prepare a desalting column.
1.4.1 Snap open the bottom of the column and insert the column into matching collection tube. Remove and discard the column cap and centrifuge for 2 min at 1000 x g. Discard eluate.
Note: When using a fixed-angle rotor, place a mark on the side of the column that faces away from the rotor center. For all subsequent centrifugation steps, place the column in the microcentrifuge with the mark facing away from the rotor center.
1.4.2 Apply 500 μL of Conjugation Buffer and centrifuge for 2 min at 1000 x g. Discard eluate.
1.4.3 Repeat step 1.4.2 for two additional times. Add Conjugation Buffer for the third wash but wait to centrifuge until immediately prior to proceeding to step.
1.4.4 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.
1.5 Insert prepared desalting column into the flesh Collection Tube and apply the entire sample from step 1.3 to the column. If the volume is less than 60 µL, top up with Conjugation Buffer.
1.6 Centrifuge at 1000 x g for 2 min. Retain the eluate, which is the processed antibody.
2. Conjugate with SMCC activated HRP
2.1 Add the prepared antibody from step 1.6 to the SMCC Activated HRP powder and mix thoroughly. Incubate the mixture at room temperature for 1 hour and keep away from light.
2.2 Block excess free thiols
2.2.1 Add 500 µL of DMSO into the Quencher Reagent. Note: The Quencher Reagent solution must be prepared fresh before each use.
2.2.2 Add 1 µL of prepared Quencher reagent into conjugation mixture from Step 2.2 and mix completely. Incubate at room temperature for 20 min and keep away from light.
2.4 Conjugation is complete. The reaction mixture should contain mostly the HRP-antibody conjugate, with small amounts of free HRP and antibody. Unconjugated HRP should not interfere with the assay.
Storage
Add 50% glycerol to the HRP-antibody conjugate and store at -20°C protected from light.
Matters needing attention
1. The antibody or protein to be labeled should be dissolved in an amine-free buffer (e.g., MES, MOPS, HEPES, PBS) at a pH of 6.5–8.5. The solution must be free of preservatives such as sodium azide. If present, these must be removed via purification prior to the labeling reaction.
2. Before formal experimentation, allow all reagents to reach room temperature, as lower temperatures may impair conjugation efficiency.
3. Do not reuse the purification resin.
4. 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.
5. During operation, always wear a lab coat, disposable gloves, and protective equipment.
6. All products are for research use only.
| M1508746 | Component | 1 reaction | Storage | Quantity Per reaction |
| M1508746A | SMCC Activated HRP | 0.1 mg | -20℃. Store in the dark. | 0.1 mg for labeling 100 µg of antibody |
| M1508746B | Conjugation Buffer | 10mL | RT. | Prepare according to instructions |
| M1508746C | Conjugation Reagent | 2 mg | 2-8℃. Do not freeze. | Prepare according to instructions |
| M1508746D | 终止剂 | 2 mg | 2-8℃. Do not freeze. | Prepare according to instructions |
| M1508746E | DMSO | 1mL | RT. | Prepare according to instructions |
| M1508746F | 离心式脱盐柱 | 1 EA | 2-8℃. Do not freeze. | 1 EA for 1 reaction |
| M1508746G | 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 |
|---|---|---|
 C2630222 |
分析证书 | M1508746 |
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