BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
2-8°C储存,室温,-20°C储存,禁止冷冻。低温运输,禁止冷冻 。请查阅批次 COA 获取详细规格。
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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。
Maleimide快速标记试剂盒采用简便快捷的流程，可将马来酰亚胺（SMCC）基团标记到蛋白上，使其具有巯基反应性，可与含巯基(-SH)的分子如抗体等偶联，偶联物可用于蛋白质印迹、ELISA及免疫组化等应用。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。NHS酯在pH值7–8.5条件下与伯胺反应形成稳定的共价酰胺键，如图1的反应(1)，而马来酰亚胺在pH值6.5–7.5条件下与巯基基团发生反应，形成稳定的硫醚键，如图1的反应(2)。与不含环己烷环的类似试剂相比，Sulfo-SMCC间隔臂中的环己烷环可降低马来酰亚胺基团的水解速率。该特点使通过Sulfo-SMCC进行马来酰亚胺标记的蛋白能够进行冻干并储存，以便随后与含巯基分子进行偶联。本试剂盒提供足以完成10次、每次1 mg抗体标记反应所需的所有试剂和纯化柱，以及详细的分步操作指南。
图1 Maleimide快速标记试剂盒(1 mg 规格) (S1510890)标记原理图
其他所需试剂
1. 离心机
2. 用于标记的所需抗体（不含BSA或任何载体蛋白）。
3. PBS缓冲液（pH 7.2-7.4）。
使用方法
1. 准备工作
1.1 1×Conjugation Buffer配制：用PBS(pH 7.2-7.4)将10×Conjugation Buffer溶液稀释10倍，制备1×Conjugation Buffer；
1.2 Sulfo-SMCC配制(10 mg/mL)：向Sulfo-SMCC中加入400 μL 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。
1.3 用1×Conjugation Buffer溶解或稀释待标记蛋白，使浓度调整至1-10 mg/mL，若待标记蛋白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蛋白置换至1×Conjugation Buffer；
2. 确认反应比例
2.1 需根据蛋白的浓度，参考表1中的推荐比例进行标记反应。如果标记效率不理想，可酌情提高Sulfo-SMCC的比例。
表1 Sulfo-SMCC : 不同浓度蛋白的推荐摩尔比（MR）
样品类型 | 样品浓度 | 推荐摩尔比（MR） |
抗体 | 1 mg/mL | 25× - 40× |
抗体 | 2-5 mg/mL | 15× - 25× |
其他纯化蛋白 | 1–4 mg/mL | 20× |
其他纯化蛋白 | 5–10 mg/mL | 15× |
2.2 使用公式1计算所需Sulfo-SMCC的体积：
公式1
(μg protein×MR×Sulfo SMCC MW)/(protein MW×10)= 需加入的SMCC的体积(μL)
其中，µg protein 是要标记的蛋白质的质量，MR是表1中的Sulfo-SMCC与蛋白质的摩尔比，Sulfo-SMCC MW是Sulfo-SMCC的分子量（分子量为436.37 Da），protein MW 是蛋白质的分子量（单位为 Da），10是Sulfo-SMCC的浓度。例如，标记 1000 µg 的 IgG（分子量为 150,000）， 摩尔比为 25× ：
(1000×25×436.37)/（150000×10）=7.3 μL
3. SMCC标记反应
3.1 准备好待标记蛋白，根据表1和公式1计算Sulfo-SMCC用量，将两者混合后轻柔涡旋混匀；
3.2 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4的操作，完成脱盐柱的平衡。
4. 脱盐柱预平衡
4.1 将一个空的离心柱放入一个15 mL收集管中。
4.2 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)。
4.3 向脱盐柱树脂顶部加入2mL 1×Conjugation Buffer，1000 × g离心2分钟，弃去流出液；
4.4 重复步骤4.3两次，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。
4.5 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；
5. SMCC标记蛋白纯化
5.1 将准备好的脱盐柱放入新的15 mL离心管中，并将步骤3.1中的全部样品加入脱盐柱中。脱盐柱上样范围为0.4 mL-0.7 mL，若体积不足，用1×Conjugation Buffer补足；
5.2 脱盐柱1000 x g离心2分钟，收集管中的流出即为所需收集的SMCC标记蛋白。
6. 保存
6.1 立即进行下一步与带巯基蛋白(本试剂盒不提供)的交联反应。或尽快将SMCC标记的蛋白进行冻干，以待后续使用。
注意事项
1. 待标记蛋白溶液中的任何伯胺（如Tris或甘氨酸）都会干扰偶联反应，推荐使用1×Conjugation Buffer溶解待标记分子；
2. 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；
3. 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；
4. 待标记抗体体积应控制在0.4-0.7mL, 保证纯化柱的最佳去除效果；
5. 本试剂盒中的纯化填料建议一次性使用；
6. 操作过程中，务必穿戴实验服、一次性手套及防护装备；
7. 所有产品仅限于研究用途。
The Maleimide Rapid Labeling Kit uses a simple and quick process to label proteins with maleimide (SMCC) groups, conferring sulfhydryl reactivity that enables conjugation with sulfhydryl group-containing molecules such as antibodies. The conjugate can be used immediately in applications such as Western blot, ELISA, and Immunohistochemistry (IHC). Sulfo-SMCC is a crosslinker that contains sulfonated N‑hydroxysuccinimide (NHS) ester and maleimide reactive groups. NHS esters target primary amines at pH 7.0–8.5 to form amide bonds, as shown in reaction (1) of Figure 1, while maleimides react with sulfhydryl groups at pH 6.5–7.5 to form stable thioether bonds, as shown in reaction (2) of Figure 1. The cyclohexane ring in the spacer arm of Sulfo‑SMCC decreases the rate of hydrolysis of the maleimide group compared to similar reagents that do not contain this ring. This feature enables proteins that have been maleimide‑labeled with Sulfo‑SMCC to be lyophilized and stored for later use in conjugation reactions. This kit provides all necessary reagents and purification columns sufficient for 10 labeling reactions, each using 1 mg of antibody, as well as a detailed step-by-step protocol.
Required materials not supplied
1. Microcentrifuge capable of 1000 x g.
2. Desired antibody for labeling (free of BSA or any carrier protein).
3. PBS buffer (pH 7.2-7.4).
Instruction for use
1.1 Preparation of 1 × Conjugation Buffer: Dilute the 10× Conjugation Buffer solution 10-fold with PBS (pH 7.2-7.4) 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.
1.3 Dissolve or dilute the protein to be labeled with 1×Conjugation Buffer to adjust the concentration to 1–10 mg/mL. If the protein 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. Determine the Reaction Ratio
2.1 The labeling reaction should be performed according to the recommended ratios in Table 1, based on the protein concentration. If the labeling efficiency is not satisfactory, the ratio of Sulfo-SMCC may be appropriately increased.
Table 1. Recommended Sulfo-SMCC : Proteins at Different Concentrations Molar Ratios (MR)
Sample type | Sample concentration | Recommended Sulfo-SMCC molar excess |
IgG antibody | 1mg/mL | 25× - 40× |
IgG antibody | 2-5mg/mL | 15× - 25× |
Purified protein | 1–4 mg/mL | 20× |
Purified protein | 5–10 mg/mL | 15× |
2.2 Use equation 1 to calculate the appropriate volume of Sulfo-SMCC solution to use:
Equation 1
(μg protein×MR×Sulfo SMCC MW)/(protein MW×10)= μL Sulfo-SMCC to add to sample
where µg protein is the mass of protein you want to label, MR is the Sulfo-SMCC : protein molar ratio from Table 1, Sulfo-SMCC MW is the molecular weight of the Sulfo-SMCC in Da (MW 436.37 Da), protein MW is the molecular weight of your protein in Da, and 10 is the concentration of the Sulfo-SMCC solution. For example, to label 1000 µg of IgG (MW 150,000 Da), MR is 25×:
(1000×25×436.37)/（150000×10）= 7.3 μL
3. SMCC Labeling Reaction
3.1 Prepare the protein to be labeled. Calculate the required amount of Sulfo-SMCC according to Table 1 and Equation 1. Mix the two components and gently vortex to mix thoroughly.
3.2 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 labeled protein.
4. Prepare the spin column
4.1 Place an empty spin column in a 15 mL collection tube.
4.2 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).
4.3 Add 2 mL of 1×Conjugation Buffer, then centrifuge the column-tube assembly at 1000 x g for 2 minutes to equilibrate the column.
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.
4.4 Repeat step 4.3 for two additional times. Add 1×Conjugation Buffer for the third wash but wait to centrifuge until immediately prior to proceeding to step.
4.5 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.
5. Purification of SMCC-Labeled Protein
5.1 Place column into a new 15 mL collection tube and apply the entire sample from step 3.1 to the column. The loading volume range for the desalting column is 0.4 mL to 0.7 mL. If the volume is insufficient, top up with 1× Conjugation Buffer.
5.2 Centrifuge the desalting column at 1000×g for 2 minutes. The flow-through in the collection tube is the desired SMCC-labeled protein.
6. Strorage
6.1 Proceed immediately to the next step for crosslinking with the sulfhydryl-containing protein (not provided with this kit). Alternatively, lyophilize the SMCC-labeled protein as soon as possible for future use.
Important
1. Any primary amines in the IgG solution (e.g., Tris or glycine) will interfere with conjugation. It is recommended to dissolve the molecule to be labeled in 1×Conjugation Buffer.
2. 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.
3. 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.
4. The volume of the antibody to be labeled should be controlled between 0.4 mL and 0.7 mL to ensure optimal removal efficiency using the purification column.
5. The purification resin included in this kit is recommended for single use only.
6. During operation, always wear a lab coat, disposable gloves, and protective equipment.
7. All products are for research use only.
| S1510890 | 组分 | 10 reactions | 储存温度 | 每次反应用量 |
| S1510890A | Sulfo-SMCC | 4 mg | -20℃ | 4 mg可用于10次1 mg抗体的标记 |
| S1510890B | 10×Conjugation Buffer | 20 mL | 室温 | 请按说明配制 |
| S1510890C | DMSO | 1 mL | 室温 | 请按说明配制 |
| S1510890D | 纯化填料 | 30 mL | 2-8℃，禁止冷冻 | 每次反应需使用3 mL |
| S1510890E | 离心柱管 | 10 EA | 室温 | 每次反应需使用1套 |
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 |
|---|---|---|
 ZJ26F0535325 |
分析证书 | S1510890 |
| ZJ26F0535324 |
分析证书 | S1510890 |
| ZJ26F0535323 |
分析证书 | S1510890 |
| ZJ26F0535322 |
分析证书 | S1510890 |
| ZJ26F0535321 |
分析证书 | S1510890 |
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