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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。
Sulfhydryl快速标记试剂盒采用简便快捷的流程，可将游离巯基(-SH)引入任何含一级氨基（NH₂）的分子，本试剂盒标记的蛋白、抗体等能在温和的条件下快速、高效、自发地偶联带有马来酰亚胺(Maleimide)的生物分子，偶联物可用于蛋白质印迹、ELISA及免疫组化等应用。本试剂盒中采用SATA进行巯基的引入。SATA是一种含有磺化 N-羟基琥珀酰亚胺 (NHS) 酯和受保护巯基的异型双功能交联剂。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)；受保护的巯基可在偶联反应需要时再将其暴露，如图1的反应(2)，可与马来酰亚胺基团反应生成稳定的硫醚键。SATA的优势在于巯基以受保护的形式引入，经过修饰的蛋白在未进行脱保护反应前可以长期储存，在需要进行下一步交联反应时再用羟胺暴露巯基，因此偶联反应的进度可控。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 Sulfhydryl快速标记试剂盒(100µg 规格) (S1510886)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. SATA很容易受潮水解失活，保存时一定要注意保持干燥；使用试剂盒提供的无水溶剂配制成母液后，可分装后-20℃保存，两个月内有效。-80℃可以保存更长时间。
3. 盐酸羟胺容易受潮，保存时注意干燥，称量前需平衡至室温(25℃左右)再开瓶。
4. 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；
5. 脱盐柱建议一次性使用；
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。
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 SATA配制(4 mg/mL)：向SATA中加入500 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SATA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0.5-5 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SATA的比例。
表1 SATA : 不同浓度蛋白的推荐摩尔比（MR）
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2.2 使用公式1计算所需SATA的体积：
公式1
(μg protein×MR×SATA MW)/(protein MW×4)= 需加入的SATA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SATA与蛋白质的摩尔比，SATA MW是SATA的分子量（分子量为231.22 Da），protein MW 是蛋白质的分子量（单位为 Da），4是SATA的浓度。例如，标记 100 µg 的 IgG（分子量为 150,000），摩尔比为30×：
(100×30×231.22)/（150000×4）=1.16 μL
3. SATA标记反应
3.1 准备好待标记蛋白，根据表1和公式1计算SATA用量，将两者混合后轻柔涡旋混匀；
3.2 在室温(约25℃)反应30分钟或者4℃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。
4. 巯基脱保护
4.1 40 mg/mL 盐酸羟胺的配制：将盐酸羟胺平衡至室温再开瓶。称量适量的盐酸羟胺，用1×Conjugation Buffer进行溶解配制成40 mg/mL盐酸羟胺，用NaOH将pH调节至7.2。注：盐酸羟胺须现配现用。
4.2 按照步骤3.1中反应体积的1/10加入盐酸羟胺。例如，反应体积为0.5 mL，需加入50 µL 盐酸羟胺，轻微涡旋混合。
4.3 在室温(约25℃)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5的操作，完成脱盐柱的平衡。
5. 脱盐柱预平衡
5.1 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。
5.2 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
5.3 重复步骤5.2两次，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；
5.4 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；
6. 巯基标记蛋白纯化
6.1 将准备好的脱盐柱转移至新的收集管中，并将步骤4.2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；
6.2 脱盐柱1000xg离心2分钟，收集管中的流出即为所需收集的巯基标记蛋白。
7. 保存
7.1 立即进行下一步与带马来酰亚胺蛋白(本试剂盒不提供)的交联反应，或待需要进行下一步反应时再对巯基进行脱保护。
Introduction
Sulfhydryl Rapid Labeling Kit uses a simple and quick process to introduce free sulfhydryl groups (–SH) into any molecule containing primary amines (NH₂). Proteins, antibodies, and other molecules labeled with this kit can rapidly, efficiently, and spontaneously conjugate with maleimide-containing biomolecules under mild conditions. The conjugates can be used in applications such as Western blot, ELISA, and Immunohistochemistry (IHC).This kit uses SATA for the introduction of sulfhydryl groups. SATA is a heterobifunctional crosslinker that contains a sulfonated N-hydroxysuccinimide (NHS) ester and a protected sulfhydryl group. The NHS ester reacts with primary amines at pH 7–8.5 to form stable covalent amide bonds, as shown in reaction (1) of Figure 1. The protected sulfhydryl group can be exposed when needed for the conjugation reaction, as shown in reaction (2) of Figure 1, and then reacts with maleimide groups to form stable thioether bonds.The advantage of SATA is that the sulfhydryl group is introduced in a protected form. The modified protein can be stored for extended periods without deprotection. When the next crosslinking reaction is required, hydroxylamine is used to expose the sulfhydryl group, allowing the conjugation reaction progress to be controlled. 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 Sulfhydryl Rapid Labeling Kit (100 µg Scale) (S1510886) Labeling Principle Schematic
Required materials not supplied
1. Microcentrifuge capable of 1,000 × g.
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. SATA 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. Hydroxylamine is hygroscopic. Keep it dry during storage. Allow it to equilibrate to room temperature (approximately 25°C) before opening the bottle for weighing.
4. 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.
5. Do not reuse the purification resin.
6. During operation, always wear a lab coat, disposable gloves, and protective equipment.
7. 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 (pH 7.2-7.4) to prepare 1 × Conjugation Buffer.
1.2 Preparation of SATA (4 mg/mL): Add 500 μL of DMSO to the SATA. Vortex or pipet up and down until the solution is homogeneous. Note: If the dissolved SATA is not used immediately, aliquot the remaining solution and store at -20°C for up to two months. Storage at -80°C allows for even longer preservation.
1.3 Dissolve or dilute the protein to be labeled with 1 × Conjugation Buffer to adjust the concentration to 0.5–5 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 SATA may be appropriately increased.
Table 1. Recommended SATA : Proteins at Different Concentrations Molar Ratios (MR)
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2.2 Use equation 1 to calculate the appropriate volume of SATA solution to use:
Equation 1
(μg protein×MR×SATA MW)/(protein MW×4)= μL SATA to add to sample
where µg protein is the mass of protein you want to label, MR is the SATA : protein molar ratio from Table 1, SATA MW is the molecular weight of the SATA in Da (MW 231.22 Da), protein MW is the molecular weight of your protein in Da, and 4 is the concentration of the SATA solution. For example, to label 100 µg of IgG (MW 150,000 Da), MR is 30×:
(100×30×231.22)/（150000×4）=1.16 μL
3. SATA Labeling Reaction
3.1 Prepare the protein to be labeled. Calculate the required amount of SATA 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.
4. De-protect the latent sulfhydryl
4.1 Preparation of Hydroxylamine HCl (40 mg/mL): Allow the Hydroxylamine HCl to equilibrate to room temperature before opening the bottle. Weigh an appropriate amount of Hydroxylamine HCl and dissolve it in 1 × Conjugation Buffer to prepare a 40 mg/mL Hydroxylamine HCl solution. Adjust the pH to 7.2 using NaOH. Note: Hydroxylamine HCl must be prepared fresh immediately before use.
4.2 Add Hydroxylamine HCl at 1/10 of the reaction volume from step 3.1. For example, for a reaction volume of 0.5 mL, add 50 µL of hydroxylamine hydrochloride and mix gently by vortexing.
4.3 Incubate at room temperature (approximately 25°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 5 below, to prepare a spin column for the purification of the labeled protein.
5. Prepare the spin column
5.1 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.
5.2 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.
5.3 Repeat step 5.2 for two additional times. Add 1 × Conjugation Buffer for the third wash but wait to centrifuge until immediately prior to proceeding to step.
5.4 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.
6. Purification of sulfhydryl-Labeled Protein
6.1 Insert prepared desalting column into the new Collection Tube and apply the entire sample from step 4.2 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.
6.2 Centrifuge the desalting column at 1000 × g for 2 minutes. The flow-through in the collection tube is the desired sulfhydryl-labeled protein.
7. Strorage
7.1 Proceed immediately to the next step of conjugation with maleimide-containing protein (not provided with this kit), or perform deprotection of the sulfhydryl groups when the next reaction is required.
| S1510886 | 组分 | 10 reactions | 储存温度 | 每次反应用量 |
| S1510886A | SATA | 2 mg | -20℃. | 2 mg可用于10次100 μg抗体的标记 |
| S1510886B | 10×Conjugation Buffer | 3 mL | 室温 | 请按说明配制 |
| S1510886C | DMSO | 1 mL | 室温 | 请按说明配制 |
| S1510886D | 盐酸羟胺 | 50 mg | 室温，防潮，避光 | 请按说明配制 |
| S1510886E | 离心式脱盐柱 | 10 EA | 2-8℃，禁止冷冻 | 每次反应需使用1套 |
| S1510886F | 收集管 | 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 |
|---|---|---|
 ZJ26F0535309 |
分析证书 | S1510886 |
| ZJ26F0535310 |
分析证书 | S1510886 |
| ZJ26F0535311 |
分析证书 | S1510886 |
| ZJ26F0535312 |
分析证书 | S1510886 |
| ZJ26F0535313 |
分析证书 | S1510886 |
| ZJ26F0535314 |
分析证书 | S1510886 |
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