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,避光,室温,禁止冷冻。低温运输,禁止冷冻 。请查阅批次 COA 获取详细规格。
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
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。
APC标记试剂盒采用简便快捷的流程，实现抗体的APC标记。该试剂盒提供标记最多1 mg抗体所需的所有试剂、纯化填料和离心柱管，以及详细的分步操作指南。
APC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APC形成共价键，从而快速实现APC与抗体的偶联。
图1 APC标记试剂盒 (1 mg) (A1491979) 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 APC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 配制Conjugation Reagent溶液（2 mg/mL）：向Conjugation Reagent中加入1 mL的纯水，混匀。
注意：Conjugation Reagent溶液须现用现配。
1.3 每1 mg抗体溶液中加入9.17 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 将一个空的离心柱放入一个15 mL收集管中。
1.4.2 通过反复倒置试剂瓶重悬纯化树脂。然后取2.0 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)。
1.4.3 向脱盐柱树脂顶部加入2 mL Conjugation Buffer，1000 × g离心2分钟，弃去流出液。
1.4.4 重复步骤1.4.3两次，第三次洗涤时加入Conjugation Buffer，直到进入下一步骤时再进行离心。
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。
1.4.5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。
1.5 将准备好的脱盐柱放入配套的离心管中，并将步骤1.3中的全部样品加入脱盐柱中。注：若体积不足260 µL，用Conjugation Buffer补足。
1.6 1000 × g离心2 min，保留洗脱液，得到巯基化的IgG。
2. 与APC进行偶联
2.1 配制APC溶液：向SMCC Activated APC中加入400 μL纯水，轻微涡旋直至全部溶解。注：复溶的SMCC Activated APC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 APC溶液中，室温避光孵育1 h。
2.3 阻断过量的游离巯基
2.3.1 配制终止剂（4 mg/mL）：向终止剂中加入500 µL DMSO，涡旋混匀。
2.3.2 向步骤2.2制备的偶联混合液中加入4.17 µL 终止剂，充分混合，室温避光孵育20 min。
2.4 偶联反应已完成。反应混合物中应主要含有APC-抗体偶联物，并含有少量游离的APC和抗体。未偶联的APC不应干扰检测。
偶联物保存
1. 将APC-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. 本试剂盒中的纯化填料建议一次性使用。
4. 待标记抗体体积应控制在260µL - 450µL，保证纯化柱的最佳去除效果。
5. 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。
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。
APC Conjugation Kit uses a simple and quick process for APC conjugation of antibodies. The kit provides all the reagents, purification resin and empty spin columns needed to label to 1 mg of antibody, as well as a detailed step-by-step protocol.The APC 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 APC and sulfhydryl group on the antibody form a covalent bond during conjugation.
Fig. 1 APC Conjugation Kit (1 mg) (A1491979) 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 APC.
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 Prepare Conjugation Reagent solution (2 mg/mL): 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 9.17 µL of prepared Conjugation Reagent per 1 mg of antibody solution, briefly vortex to mix.
1.4 During the incubation prepare a desalting column.
1.4.1 Place an empty spin column in a 15 mL collection tube.
1.4.2 Suspend the purification resin by repeated inversion of the reagent bottle. Then add 2.0 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).
1.4.3 Add 2 mL of Conjugation Buffer, then centrifuge the column-tube assembly at 1000 x g for 2 minutes to equilibrate the column.
1.4.4 Repeat step 1.4.3 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.
1.4.5 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 the matching Collection Tube and apply the entire sample from step 1.3 to the column.
Note: If the volume is less than 260 µL, top up with Conjugation Buffer.
1.6 Centrifuge at 1000 × g for 2 min. Retain the eluate, which is the processed antibody.
2. Conjugate with SMCC activated APC
2.1 Prepare APC solution: Add 400 μL of purified water to SMCC Activated APC. Vortex gently until the solution is homogenous. Note: The Reconstituted SMCC Activated APC solution must be prepared fresh before each use.
2.2 Add the prepared antibody from step 1.6 to the prepared APC solution. Incubate the mixture at room temperature for 1 hour and keep away from light.
2.3 Block excess free thiols
2.3.1 Prepare Quencher Reagent solution (4 mg/mL): Add 500 µL of DMSO into the Quencher Reagent.
Note: The Quencher Reagent solution must be prepared fresh before each use.
2.3.2 Add 4.17 µL of prepared Quencher reagent into conjugation mixture from Step 2.2 and mix completely. Incubate at room temperature for 20 minutes and keep away from light.
2.4 Conjugation is complete. The reaction mixture should contain mostly the APC-antibody conjugate, with small amounts of free APC and antibody. Unconjugated APC should not interfere with the assay.
Storage
1. Store the APC-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. The purification resin included in this kit is recommended for single use only.
4. The volume of the antibody to be labeled should be controlled between 260 µL and 450 µL to ensure optimal removal efficiency using the purification column.
5. Fluorescent dyes are prone to quenching. Please store and use them in the dark to slow down fluorescence quenching.
6. During operation, always wear a lab coat, disposable gloves, and protective equipment.
7. All products are for research use only.
| A1491979 | 组分 | 物理外观 | 1 reaction | 储存温度 | 每次反应用量 |
| A1491979A | SMCC Activated APC | 蓝色冻干固体 | 1 vial | 2-8℃，避光，禁止冷冻 | 1管可用于标记最多1mg的抗体 |
| A1491979B | Conjugation Buffer | 无色透明液体 | 20mL | 室温 | 请按说明配制 |
| A1491979C | Conjugation Reagent | 白色固体 | 2 mg | 2-8℃，禁止冷冻 | 请按说明配制 |
| A1491979D | 终止剂 | 白色固体 | 2 mg | 2-8℃，禁止冷冻 | 请按说明配制 |
| A1491979E | DMSO | 无色透明液体 | 1mL | 室温 | 请按说明配制 |
| A1491979F | 纯化填料 | 白色微珠混悬液 | 2 mL | 2-8℃，禁止冷冻 | 每次反应需使用2mL |
| A1491979G | 离心柱管 | 透明管体 | 1 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 |
|---|---|---|
 ZJ26F0737068 |
分析证书 | A1491979 |
| ZJ26F0737067 |
分析证书 | A1491979 |
| ZJ26F0737066 |
分析证书 | A1491979 |
| ZJ26F0737065 |
分析证书 | A1491979 |
| ZJ26F0737064 |
分析证书 | A1491979 |
| ZJ26F0737063 |
分析证书 | A1491979 |
| ZJ26F0737062 |
分析证书 | A1491979 |
危险品化学品经营许可证（带存储）