BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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。
抗体-多肽偶联试剂盒(SMCC法)采用简便快捷的流程，可将马来酰亚胺(SMCC)基团标记到抗体上，使其具有巯基反应性，可与含巯基(-SH)的多肽偶联，所得偶联物可刺激机体产生免疫应答，制备靶向半抗原的抗体。多肽免疫原性极低，无法直接刺激机体产生特异性抗体，且在体内易被快速代谢降解，无法有效激发免疫反应。因此需将其与大分子载体蛋白偶联，依靠载体蛋白的强免疫原性，辅助多肽激活机体特异性免疫应答，保障免疫效果和抗体制备成功率。
Sulfo-SMCC是一种含有磺化N-羟基琥珀酰亚胺(NHS)酯和马来酰亚胺基团的异型双功能交联剂，可与伯胺和半胱氨酸/巯基分子的共价偶联。用户需自备抗体与含巯基多肽。试剂盒提供Sulfo-SMCC、偶联所需的所有试剂、纯化填料和离心柱管，以及详细的分步操作指南。本试剂盒包含两种规格，可支持1次或5次独立偶联反应，每次可标记2mg抗体。
图1 抗体-多肽偶联试剂盒(SMCC法) (A1509086)标记原理图
产品组份
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其他所需试剂
1. 离心机。
2. 用于偶联的抗体和含巯基多肽。
3. PBS缓冲液（pH 7.2-7.4）。
注意事项
1. 本方案为通用流程，每次可支持2mg抗体的偶联反应，适用于绝大多数多肽；需根据多肽分子量、空间结构优化条件以提升偶联效率。
2. 体系中多肽摩尔量过量可保证偶联充分；通常多肽与抗体等质量投料即可实现摩尔过量。
3. 反应体系不能含有任何伯胺（如Tris或甘氨酸），这些物质会干扰偶联反应。
4. Sulfo-SMCC很容易受潮水解失活，保存时一定要注意封口保持干燥；使用DMSO配制成母液后，可分装后-20℃保存两个月。-80℃可以保存更长时间。
5. 反应体系添加过DMSO时，平衡柱子的纯化缓冲液需加入同等比例DMSO，避免脱盐过程蛋白析出。
6. 纯化填料建议一次性使用。
7. 在正式实验前，需将试剂恢复至室温，在使用前将试剂进行离心，较低的温度和离心管壁试剂残留均会影响偶联效果。
8. 操作过程中，务必穿戴实验服、一次性手套及防护装备。
9. 所有产品仅限于研究用途。
使用方法
1. 准备工作
1.1 配制1 × Conjugation Buffer：用PBS将10 × Conjugation Buffer溶液稀释10倍，制备1 × Conjugation Buffer。
1.2 配制Sulfo-SMCC(10 mg/mL)：向Sulfo-SMCC中加入400 μL的DMSO，用涡旋振荡器振荡或用移液器上下吹吸，直至溶液混合均匀。
注意：溶解后的Sulfo-SMCC若不立即用完，将剩余的分装后-20℃保存，2个月内有效。
2.SMCC活化抗体
2.1 准备抗体溶液（10 mg/mL）：向2 mg抗体中加入200 μL 1×Conjugation Buffer。若待标记抗体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1×Conjugation Buffer。
2.2 向准备好的抗体溶液中加入11.7 μL Sulfo-SMCC溶液，在室温(约25ºC)反应30分钟或者4ºC反应2小时，由于样品体积较小，推荐使用微型涡旋混合器，1500 r。
注意：反应进行期间，可提前进行步骤3的操作，完成脱盐柱的平衡。
3. 脱盐柱预平衡
3.1 将一个空的离心柱放入一个15 mL收集管中。
3.2 通过反复倒置试剂瓶重悬纯化树脂。然后取1.0 mL悬液加入柱中，让树脂沉降。让柱缓冲液依靠重力从柱中排出。初始时，可能需要施加一些压力使最初的几滴缓冲液流出。以1000 × g离心柱子2分钟，弃去储存缓冲液，将柱子放回同一个收集管中。
注意：纯化树脂以20%乙醇的悬液形式提供，树脂与乙醇的体积比为3:1(v/v)。
3.3 向脱盐柱树脂顶部加入1 mL 1×Conjugation Buffer，1000 × g离心2分钟，弃去流出液。
3.4 重复步骤3.3两次，第三次洗涤时加入1 × Conjugation Buffer，直到进入下一步骤时再进行离心。
注意：使用定角转子时，在柱子背对转子中心的一侧做标记。在后续所有离心步骤中，将柱子放入离心机时，标记面应背对转子中心。
3.5 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。
4. SMCC活化抗体纯化
4.1 将准备好的脱盐柱放入15 mL离心管中，并将步骤2.2中的全部样品加入脱盐柱中。
4.2 脱盐柱1000 × g离心2分钟，收集管中的流出即为所需收集的SMCC活化抗体。
5. 抗体-多肽偶联
5.1 配制多肽溶液（5 mg/mL）：取2 mg多肽，溶于400 μL 1×Conjugation Buffer。
注意：部分多肽水溶性差，可用DMSO助溶，DMSO在终反应体系中占比＜30%；过高浓度DMSO会造成抗体不可逆变性。
5.2 将400 μL多肽溶液全部加入到步骤4.2的SMCC活化抗体中，室温避光孵育2 h。
注意：反应进行期间，可提前进行步骤6的操作，完成脱盐柱的平衡。
6. 脱盐柱预平衡
6.1 将一个空的离心柱放入一个15 mL收集管中。
6.2 通过反复倒置试剂瓶重悬纯化树脂。然后取3.0 mL悬液加入柱中，让树脂沉降。让柱缓冲液依靠重力从柱中排出。初始时，可能需要施加一些压力使最初的几滴缓冲液流出。以1000 × g离心柱子2分钟，弃去储存缓冲液，将柱子放回同一个收集管中。
注意：纯化树脂以20%乙醇的悬液形式提供，树脂与乙醇的体积比为3:1(v/v)。
6.3 向脱盐柱树脂顶部加入2 mL PBS，1000 × g离心2分钟，弃去流出液。
注意：若偶联物短期1周内使用：可使用PBS完成脱盐；如需冻存，必须使用试剂盒配套的纯化缓冲液，可避免冻融过程蛋白失活析出。
6.4 重复步骤6.3两次，第三次洗涤时加入PBS，直到进入下一步骤时再进行离心。
注意：使用定角转子时，在柱子背对转子中心的一侧做标记。在后续所有离心步骤中，将柱子放入离心机时，标记面应背对转子中心。
6.5 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。
7. 抗体-多肽偶联物纯化
7.1 将准备好的脱盐柱放入新的15 mL离心管中，并将步骤5.2中的全部样品加入脱盐柱中。
注意：若反应出现沉淀，离心收集上清单独纯化，沉淀留存；纯化完成后将沉淀与纯化液合并使用。
7.2 脱盐柱1000 x g离心2分钟，收集管中的即为纯化抗体-多肽偶联物。
8. 保存
8.1 纯化后可直接用于动物免疫；若存放超过数日，无菌过滤后置于无菌容器，4℃短期保存或分装-20℃冻存。
Antibody Peptide Conjugation Kit (SMCC) uses a simple and quick process to label antibodies with maleimide (SMCC) groups, conferring sulfhydryl reactivity that enables conjugation with sulfhydryl group-containing small-molecules such as peptides. The obtained conjugates can stimulate immune responses in organisms for the production of peptide-targeted antibodies. Peptides exhibit extremely low immunogenicity. They cannot directly stimulate the organism to produce specific antibodies. Furthermore, they are rapidly metabolized and degraded in vivo and fail to trigger an effective immune response. Therefore, they must be conjugated to large-molecular-weight carrier proteins. Relying on the strong immunogenicity of carrier proteins, peptides can be assisted to activate the host-specific immune response, ensuring immunization performance and the success rate of antibody production.
Sulfo-SMCC is a crosslinker that contains sulfonated N-hydroxysuccinimide (NHS) ester and maleimide reactive groups, enabling covalent conjugation with primary amines and cysteine-/thiol-containing molecules. The user supplies the antibody and peptide. This kit contains Sulfo-SMCC, all reagents required for conjugation, purification resin, empty spin columns, and a detailed step-by-step protocol. This kit is suitable for 1 or 5 independent reactions with 2 mg of antibody per reaction.
Fig. 1 Antibody Peptide Conjugation Kit (SMCC) (A1509086) Labeling Principle Schematic
Contents and storage
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Required materials not supplied
1. Microcentrifuge capable of 1000 × g.
2. Target antibody and thiol-containing peptides for conjugation
3. PBS buffer (pH 7.2-7.4).
Important
1. The following protocol is a general protocol for a wide variety of peptides. Optimize the protocol accordingly for the conjugation efficiencies based on the size and structure of your peptide.
2. Using a molar excess of peptide over antibody ensures efficient conjugation. In general, a reaction with equal mass amounts of peptide and antibody will achieve sufficient molar excess.
3. The reaction system must not contain any primary amines (e.g., Tris or glycine), as these substances will interfere with the conjugation reaction.
4. Sulfo-SMCC is easily inactivated by moisture-induced hydrolysis. Care must be taken to seal the container tightly and keep it dry during storage. After preparing the stock solution in DMSO, it can be aliquoted and stored at -20°C for up to two months. Storage at -80°C allows for even longer preservation.
5. If DMSO was used in the conjugation, prepare the Purification Buffer Salts with the same percentage of DMSO used for the conjugation. This minimizes precipitation in the column during desalting.
6. Do not reuse the purification resin.
7. Before formal experimentation, the reagents must be returned to room temperature and centrifuged before use. Both lower temperatures and reagent residue on the centrifuge tube walls may impair conjugation efficiency.
8. During operation, always wear a lab coat, disposable gloves, and protective equipment.
9. All products are for research use only.
Instruction for use
1.1 Preparation of 1 × Conjugation Buffer: Dilute the 10 × Conjugation Buffer solution 10-fold with PBS to prepare 1 × Conjugation Buffer.
1.2 Preparation of Sulfo-SMCC (10 mg/mL): Add 400 μL of DMSO to the Sulfo-SMCC. Vortex or pipet up and down until the solution is homogeneous.
Note: If the dissolved Sulfo-SMCC is not used immediately, aliquot the remaining solution and store at -20°C for up to two months.
2. SMCC-Activated Antibody Preparation Protocol
2.1 Preparation of antibody solution (10 mg/mL): Add 200 μL of 1 × Conjugation Buffer to 2 mg of antibody. If the antibody to be labeled is not in a salt-free state or is already in a solution, it is necessary to exchange the buffer into 1 × Conjugation Buffer by concentration or dialysis.
2.2 Add 11.7 μL Sulfo-SMCC solution to the prepared antibody solution, Incubate at room temperature (approximately 25°C) for 30 minutes or at 4°C for 2 hours. Due to the small sample volume, a micro-vortex mixer is recommended at 1500 rpm.
Note: During the incubation period, proceed to steps 3 below, to prepare a spin column for the purification of the activated antibody.
3. Prepare the spin column
3.1 Place an empty spin column in a 15 mL collection tube.
3.2 Suspend the purification resin by repeated inversion of the reagent bottle. Then add 1.0 mL of the suspension into the column and allow the resin to settle. Allow the column buffer to drain from the column by gravity. Initially, some pressure may be required to cause the first few drops of buffer to elute. Centrifuge the column at 1000 × g for 2 minutes, discard the storage buffer and return column to the same collection tube.
Note: The purification resin is supplied as a slurry in 20% ethanol, with a resin-to-ethanol volume ratio of 3:1 (v/v).
3.3 Add 1 mL of 1 × Conjugation Buffer, then centrifuge the column-tube assembly at 1000 × g for 2 minutes to equilibrate the column.
3.4 Repeat step 3.3 for two additional times. Add 1 × Conjugation Buffer for the third wash but wait to centrifuge until immediately prior to proceeding to step.
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.
3.5 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.
4. Purification of SMCC-Activated Antibody
4.1 Place column into a 15 mL collection tube and apply the entire sample from step 2.2 to the column.
4.2 Centrifuge the desalting column at 1000 × g for 2 minutes. The flow-through in the collection tube is the desired SMCC-activated antibody.
5. Antibody Peptide Conjugation Protocol
5.1 Prepare peptide solution (5 mg/mL): Dissolve 2 mg peptide in 400 µL 1×Conjugation Buffer .
Note: For haptens with limited solubility, use < 30% DMSO in the final conjugation solution. Using a higher concentration of DMSO might irreversibly denature the carrier protein.
5.2 Add the 400 μL of peptide solution to the 200 μL prepared antibody solution. Incubate at room temperature for 2 hours.
Note: During the incubation period, proceed to steps 6 below, to prepare a spin column for the purification of the labeled protein.
6. Prepare the spin column
6.1 Place an empty spin column in a 15 mL collection tube.
6.2 Suspend the purification resin by repeated inversion of the reagent bottle. Then add 3.0 mL of the suspension into the column and allow the resin to settle. Allow the column buffer to drain from the column by gravity. Initially, some pressure may be required to cause the first few drops of buffer to elute. Centrifuge the column at 1000 × g for 2 minutes, discard the storage buffer and return column to the same collection tube.
Note: The purification resin is supplied as a slurry in 20% ethanol, with a resin-to-ethanol volume ratio of 3:1 (v/v).
6.3 Add 2 mL of PBS Buffer, then centrifuge the column-tube assembly at 1000 × g for 2 minutes to equilibrate the column.
Note: If the conjugate is to be used for injection within one week, PBS may be used for purification. If the conjugate will be frozen, use the Purification Buffer for purification, which will preserve the product during freeze-thaw cycles.
6.4 Repeat step 6.3 for two additional times. Add PBS for the third wash but wait to centrifuge until immediately prior to proceeding to step.
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.
6.5 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.
7. Purification of Antibody-Peptide Conjugate
7.1 Place column into a new 15 mL collection tube and apply the entire sample from step 5.2 to the column.
Note: If a precipitate formed during conjugation, centrifuge the precipitated material, collect the supernatant and save the precipitate. Purify only the supernatant. Combine the purified conjugate to the precipitate.
7.2 Centrifuge the column-tube assembly at 1000 × g for 2 minutes. The purified antibody-peptide conjugates are in the collection tube.
8. Strorage
8.1 The peptide-carrier conjugate can now be used for immunization. If the peptide-carrier conjugate is to be stored for more than a few days, sterile filter the conjugate and store in a sterile container at 4℃ or -20℃
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 1 reaction | 5 reactions | 储存 | 单次测试使用量 |
| A1509086NT | 抗体-多肽偶联试剂盒(SMCC法) (常温组分) | A1509086A | 10×偶联缓冲液 | 无色透明液体 | 2 mL | 10 mL | 室温 | 请按说明配制 |
| | | A1509086B | 纯化缓冲液 | 无色透明液体 | 10 mL | 50 mL | 室温 | 请按说明配制 |
| | | A1509086C | DMSO | 无色透明液体 | 0.5 mL | 0.5 mL | 室温 | 请按说明配制 |
| | | A1509086D | 离心柱管 | 透明管体 | 2 EA | 10 EA | 室温 | 每次反应需使用2套 |
| A1509086WT | 抗体-多肽偶联试剂盒(SMCC法) (2-8℃组分) | A1509086E | 纯化填料 | 白色微珠混悬液 | 4 mL | 20 mL | 2-8℃，禁止冷冻 | 每次反应需使用4mL |
| A1509086DT | 抗体-多肽偶联试剂盒(SMCC法) (-20℃组分) | A1509086F | Sulfo-SMCC | 白色粉末 | 4 mg | 4 mg | -20℃. | 请按说明配制 |
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0939427 |
分析证书 | A1509086 |
| ZJ26F0939426 |
分析证书 | A1509086 |
| ZJ26F0939425 |
分析证书 | A1509086 |
| ZJ26F0939424 |
分析证书 | A1509086 |
| ZJ26F0939423 |
分析证书 | A1509086 |
| ZJ26F0939422 |
分析证书 | A1509086 |
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