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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。
PE-Cy5标记试剂盒采用简便快捷的流程，实现抗体的PE-Cy5标记。该试剂盒提供标记最多300 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。
PE-Cy5标记试剂盒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PE-Cy5形成共价键，从而快速实现PE-Cy5与抗体的偶联。
图1 PE-Cy5标记试剂盒（P1491952）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 PE-Cy5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300 µg抗体溶液中加入2.8 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新的收集管中，并将步骤1.3中的全部样品加入脱盐柱中；
1.6 1000 × g离心2 min，保留洗脱液，得到巯基化的IgG；
2. 与PE-Cy5进行偶联
2.1 配制PE-Cy5溶液（5 mg/mL）——向SMCC Activated PE-Cy5中加入200 μL纯水，轻微涡旋直至全部溶解。 注：复溶的SMCC Activated PE-Cy5溶液须现用现配；
2.2 将1.6中巯基化的抗体直接加入SMCC Activated PE-Cy5溶液中，室温避光孵育1 h；
2.3 阻断过量的游离巯基
偶联物保存
1. 将PE-Cy5-抗体偶联物 以>0.5 mg/mL浓度储存，或添加1-10 mg/mL牛血清白蛋白（BSA）作为稳定剂；
2. 加入防腐剂（如0.05% Proclin 300）；
3. 抗体偶联物溶液可避光于4°C保存两个月，严禁冷冻；
4. 若溶液出现聚集现象，需离心30秒后仅取上清液使用。
注意事项
1. IgG溶液中的任何伯胺（如Tris或甘氨酸）都会干扰偶联反应，使用前可将样品透析或脱盐至适宜缓冲液中；
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. 荧光染料均存在淬灭问题，请尽量注意避光，以减缓荧光的淬灭；
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。
PE-Cy5 Conjugation Kit uses a simple and quick process for PE-Cy5 conjugation of antibodies. The kit provides all the reagents, purification columns needed to label up to 300 μg of antibody, as well as a detailed step-by-step protocol.
The PE-Cy5 Conjugation Kit 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 PE-Cy5 and sulfhydryl group on the antibody form a covalent bond during conjugation.
Fig. 1 PE-Cy5 Conjugation Kit (P1491952) 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 PE-Cy5.
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 2.8 µL of prepared Conjugation Reagent per 300 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 a fresh Collection Tube and apply the entire sample from step 1.3 to the column.
1.6 Centrifuge at 1000 x g for 2 min. Retain the eluate, which is the activated antibody.
2. Conjugate with SMCC activated PE-Cy5
2.1 Prepare PE-Cy5 solution (5 mg/mL)—Add 200 μL of purified water to SMCC Activated PE-Cy5. Vortex gently until the solution is homogenous. Note: The Reconstituted SMCC Activated PE-Cy5 solution must be prepared fresh before each use.
2.2 Add the prepared antibody from step 1.6 to the prepared PE-Cy5 solution. Incubate the mixture at room temperature for 1 hour and keep away from light.
2.3 Block excess free thiols
2.3.1 Add 500 µL of DMSO into the Quencher Reagent. Note: The Quencher Reagent solution must be prepared fresh before each use.
Add 2.5 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 PE-Cy5-antibody conjugate, with small amounts of free PE-Cy5 and antibody. Unconjugated PE-Cy5 should not interfere with the assay.
Storage
1. Store the PE-Cy5-antibody conjugate at concentration > 0.5 mg/mL or add 1-10 mg/mL of BSA as a stabilizer.
2. Add preservative (e.g. 0.05% proclin 300).
3. Store the conjugate solution at 4°C in the dark for two months. DO NOT FREEZE.
4. If there is aggregation in the conjugate solution, centrifuge briefly for 30 sec and use the supernatant only.
Matters needing attention
1. Any primary amines in the IgG solution (e.g., Tris or glycine) will interfere with conjugation. If necessary, dialyze or desalt samples into an appropriate buffer, before use.
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. The reactive dyes should be protected from prolonged exposure to light.
5. During operation, always wear a lab coat, disposable gloves, and protective equipment.
6. All products are for research use only.
| P1491952 | Component | 1 reaction | Storage | Quantity Per reaction |
| P1491952A | SMCC Activated PE-Cy5 | 1 mg | 2-8℃. Store in the dark. Do not freeze. | 1 mg for labeling up to 300 μg of antibody |
| P1491952B | Conjugation Buffer | 10mL | RT. | Prepare according to instructions |
| P1491952C | Conjugation Reagent | 2 mg | 2-8℃. Do not freeze. | Prepare according to instructions |
| P1491952D | 终止剂 | 2 mg | 2-8℃. Do not freeze. | Prepare according to instructions |
| P1491952E | DMSO | 1mL | RT. | Prepare according to instructions |
| P1491952F | 离心式脱盐柱 | 1 EA | 2-8℃. Do not freeze. | 1 EA for 1 reaction |
| P1491952G | 收集管 | 1 EA | RT. | 1 EA for 1 reaction |
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