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100 μg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(100µg 规格) (S1510889)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）。
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. 脱盐柱建议一次性使用；
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。
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200 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 | 25X-40X |
抗体 | 2-5 mg/mL | 15X-25X |
其他纯化蛋白 | 1–4 mg/mL | 20X |
其他纯化蛋白 | 5–10 mg/mL | 15X |
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 100 µg 的 IgG（分子量为 150,000）：(100×25×436.37)/（150000×10）=0.73 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 松开脱盐柱盖子，折断柱子下端堵头，将脱盐柱置于配套的2mL收集管中。1000×g离心2分钟，丢弃离心下来的保存溶液，并将脱盐柱放回收集管中。
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。
4.2 向脱盐柱树脂顶部加入500μL 1×Conjugation Buffer，轻轻拧上脱盐柱上盖，1000×g离心2分钟，丢弃溶液
4.3 重复步骤4.2两次，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；
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；
5. SMCC标记蛋白纯化
5.1 将准备好的脱盐柱转移至新的收集管中，并将步骤3.1中的全部样品加入脱盐柱中，盖上脱盐柱盖子，注意盖子轻轻拧上即可。脱盐柱上样范围为70µL-120µL，若体积不足，用1×Conjugation Buffer补足；
5.2 脱盐柱1000xg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。
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 100 μg of antibody, as well as a detailed step-by-step protocol.
Fig. 1 Maleimide Rapid Labeling Kit (100 µg Scale) (S1510889) Labeling Principle Schematic
Required materials not supplied
1. Microcentrifuge capable of 1,000 xg.
2. Desired antibody for labeling (free of BSA or any carrier protein).
3. PBS buffer (pH 7.2-7.4).
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. Do not reuse the purification resin.
5. During operation, always wear a lab coat, disposable gloves, and protective equipment.
6. All products are for research use only.
Instruction for use
1. Preparations
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 200 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 | 25X-40X |
IgG antibody | 2-5mg/mL | 15X-25X |
Purified protein | 1–4 mg/mL | 20X |
Purified protein | 5–10 mg/mL | 15X |
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 100 µg of IgG (MW 150,000 Da):
(100×25×436.37)/（150000×10）=0.73 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 Loosen the cap on a spin column, twist the tab off of the bottom, then place the column into a collection tube. Centrifuge the desalting column assembly at 1,000 x g for 2 minutes to remove the storage solution. Discard the flowthrough, then place the column back into the collection tube.
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.
4.2 Add 500 μL of 1×Conjugation Buffer, then centrifuge the desalting column assembly at 1,000 x g for 2 minutes to equilibrate the column.
4.3 Repeat step 4.2 for two additional times. Add 1×Conjugation Buffer for the third wash but wait to centrifuge until immediately prior to proceeding to step.
4.4 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 Insert prepared desalting column into the new Collection Tube and apply the entire sample from step 3.1 to the column. Cap the desalting column, taking care to screw the cap on gently. The loading volume range for the desalting column is 70 µL to 120 µL. 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.
ZJ26F0434201
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