阿拉丁Azide蛋白标记试剂盒（5mg 规格）（A1510881）可以快速高效的在含有伯胺基团的目标蛋白中引入叠氮基团，随后进行点击化学反应。该点击化学反应可通过铜(I)或钌催化进行，也可与特定类型的炔烃伙伴（例如DBCO）通过环张力催化反应实现。该试剂盒提供的N3-PEG4-NHS酯，这是一种异双功能交联剂，易于与伯胺反应，形成共价连接的叠氮基团，这些基团可进一步与DBCO标记或炔基标记的偶联伙伴反应，生成多种生物共轭物。该试剂盒包含用于叠氮标记和纯化10份2mL样品（浓度范围为1-10 mg/mL）的试剂。标记和纯化过程最快可在90分钟内完成。该试剂盒针对分子量在20 kDa至150 kDa之间的蛋白质标记进行了优化。
阿拉丁Azide蛋白标记试剂盒的特点：
- 多功能——标记体积为0.5-5mL的蛋白质（20–150 kDa）
- 异型双功能—对胺基和炔基或DBCO具有反应活性
- 稳定性——PEG链增强了水溶性并减少反应的空间位阻
- 便捷性——标记和纯化最快可在90分钟内完成，实际操作时间仅需30分钟
其他所需试剂
1. 变速台式离心机。
2. 5毫升离心管。
3. 待标记的目标蛋白（不含BSA或任何载体蛋白）。
4. PBS缓冲液（pH 7.2-7.4）。
5. 紫外-可见分光光度计。
注意事项
1. 纯化后的蛋白质应置于不含伯胺（例如，叠氮化物、铵离子、Tris、甘氨酸、乙醇胺、三乙胺、谷胱甘肽）或咪唑的缓冲液中。所有这些物质都会显著抑制蛋白标记。
2. 不纯的蛋白质或用牛血清白蛋白（BSA）或明胶稳定的蛋白质将无法被高效标记。
3. 交联剂对湿气敏感。打开前应将试剂管平衡至室温，以防止试剂管凝结水分。使用试剂盒中提供的无水溶剂配制储备液后，可将其分装并在-20°C下储存，在此条件下可稳定保存两个月。在-80°C下储存可保存更长时间。
4. 离心脱盐柱的上样体积必须在2–4 mL之间，以确保最佳性能。超出此范围的上样体积可能导致蛋白质回收率降低或未偶联试剂去除不彻底。
5. 请勿重复使用离心脱盐柱。
使用说明
1. 制备蛋白质样品
1.1 将目标蛋白溶解于不含胺、铵离子或叠氮化物的缓冲液（pH 7-9）中，因为这些物质会干扰标记反应。如有必要，在标记前将目标蛋白通过透析或缓冲液置换的方式更换到合适的缓冲液中，例如PBS（1X）。
1.2 在PBS溶液中制备浓度为1-10 mg/mL的蛋白质样品。最终体积应≤4mL。
2. 计算N3-PEG4-NHS体积
N3-PEG4-NHS的最佳用量取决于所需的标记程度。通常，较高的蛋白质浓度或表面具有更多可接触伯胺（例如赖氨酸残基）的蛋白质，需要较少的N3-PEG4-NHS 即可达到所需的标记密度。关于Azide:蛋白质比例的建议，请参见表1。
| 蛋白浓度范围 | 摩尔比 (Azide:Protein) |
| 1.0-5.0 mg/mL | 50:1-20:1 |
| 5.0-10 mg/mL | 20:1-10:1 |
表1. N3-PEG4-NHS ester 与目标蛋白的推荐摩尔比
2.1 计算摩尔比为X：1所需的N3-PEG4-NHS试剂毫摩尔数：
2.2 计算需添加到反应体系中的25mM N3-PEG4-NHS体积：
2.3 示例：对于2mL的2 mg/mL IgG（分子量 = 150,000 g/mol），摩尔比为20:1时，需向制备好的样品中加入20.7 μL的25 mM N3-PEG4-NHS试剂。
3. 蛋白质标记步骤
3.1 将N3-PEG4-NHS酯平衡至室温。
3.2 使用前，配制交联剂。向N3-PEG4-NHS的试剂管中加入1mL DMSO，得到25mM的储备液。涡旋或用移液器反复吹吸直至溶液均一。
3.3 量取计算体积的25 mM N3-PEG4-NHS试剂加入制备好的蛋白溶液中。
3.4 将反应混合物在室温下孵育1小时，或在冰上孵育2小时。
4. 脱盐柱预平衡
4.1 将一个空的离心柱放入一个 50mL 收集管中。
4.2 通过反复倒置试剂瓶重悬纯化树脂。然后取14mL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 1,000 × 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 通过在树脂床顶部加入10mL PBS，以 1,000 × g 离心 2 分钟来平衡柱子，弃去流出液，此步骤总共重复 3 次。此时柱子平衡完成。
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。
5. 纯化Azide标记的蛋白质
5.1 将平衡好的离心柱转移到一个新的收集管中。
5.2 小心地将整个反应混合物（2-4mL）加到柱子的中心位置。
5.3 脱盐柱1000xg离心2分钟，收集管中的流出即为所需收集的Azide标记的蛋白。
Aladdin’s Azide Protein Labeling Kit (5mg scale) (A1510881) provides a convenient method to label proteins with azide groups for use in the well-known click chemistry reaction. The click chemistry reaction proceeds by copper(I) or ruthenium catalysis or in a strain-catalyzed reaction with certain types of alkyne partners. The kit uses N3-PEG4-NHS ester, a heterobifunctional crosslinker that readily reacts with primary amines, forming covalently attached azide groups that can be further reacted with an DBCO‑labeled or alkyne-labeled coupling partner to produce diverse bioconjugates. The kit includes reagents for azide‑labeling and purification of ten 2mL samples with a concentration of 1-10 mg/mL. Labeling and purification can be performed in as little as 90 minutes. The kit is optimized for labeling proteins with molecular weights between 20 kDa and 150 kDa
’s Azide Protein Labeling Kit features include:
- Versatile—label proteins (20–150 kDa) with volumes of 0.5–5 mL
- Heterobifunctional—reactive towards amine and alkyne or DBCO
- Stability—The PEG chain enhances water solubility and reduces non-specific adsorption
- Convenient—labeling and purification can be completed in as little as 90 minutes with 30 min hands-on time
Required materials not supplied
1. Variable‑speed benchtop microcentrifuge
2. 5mL centrifuge tubes
3. Desired Protein for labeling (free of BSA or any carrier protein).
4. PBS buffer (pH 7.2-7.4).
5. UV‑Vis spectrophotometer
Matters needing attention
1. The purified proteins should be in a buffer that does not contain primary amines (for example, azide, ammonium ions, Tris, glycine, ethanolamine, triethylamine, glutathione) or imidazole. All of these substances significantly inhibit protein labeling.
2. Impure proteins or proteins stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.
3. The crosslinker is moisture‑sensitive. Equilibrate the vial to room temperature before opening to prevent moisture condensation inside the vial. After preparing the stock solution with the anhydrous solvent provided in the kit, it can be aliquoted and stored at –20 °C, where it remains stable for two months. Storage at –80 °C allows for even longer preservation.
4. The sample loading volume for Spin Desalting Columns must be between 2-4mL to ensure optimal performance. Loading volumes outside this range may lead to lower protein recovery or inefficient removal of unconjugated reagents.
5. Do not reuse Spin Desalting Columns.
Instructions for Use
1. Prepare the protein sample
1.1 Prepare the target protein in a buffer (pH 7-9) free of amines, ammonium ions, or azide as these will interfere with the labeling reaction. If necessary, dialyze or exchange the target protein into an appropriate buffer, such as PBS (1X), before labeling.
1.2 Prepare the protein sample at a concentration of 1-10 mg/mL in PBS solution. The final volume should be ≤4mL.
2. Calculations
The optimal amount of N3-PEG4-NHS will depend on the desired degree of labeling. In general, higher protein concentrations or proteins with a greater number of accessible primary amines (e.g., lysine residues) on the surface will require less N3-PEG4-NHS to achieve the desired labeling density. For recommendations on Azide:protein ratios, see Table 1.
| Protein concentration Range | Molar Ratio (Azide:Protein) |
| 1.0-5.0 mg/mL | 50:1-20:1 |
| 5.0-10 mg/mL | 20:1-10:1 |
Table 1. Recommended molar ratio of N3-PEG4-NHS to target protein
2.1 Use the following formula to calculate the amount (in millimoles) of crosslinker to add to the sample for an X:1 molar ratio.
2.2 Use the following formula to calculate the volume (in μL) of 25 mM crosslinker to add to the sample.
2.3 For 2mL of 2 mg/mL IgG (MW = 150,000 g/mol) with 20:1 molar ratio, add 20.7 μL of 25mM N3-PEG4-NHS to the prepared sample.
3. Protein labeling procedure
3.1 Equilibrate N3-PEG4-NHS ester to room temperature.
3.2 Just before use, reconstitute the crosslinker. Add 1mL of DMSO to the vial of N3-PEG4-NHS to obtain a 25 mM stock solution. Vortex or pipette up and down until the solution is homogeneous.
3.3 Add the calculated volume of 25 mM N3-PEG4-NHS to the tube.
3.4 Incubate the reaction mixture for 1 hour at room temperature or 2 hours on ice.
4. Prepare the spin column
4.1 Place an empty spin column in a 50mL collection tube.
4.2 Suspend the purification resin by repeated inversion of the reagent bottle. Then add 14 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 1,000 × 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 Equilibrate the column by adding 10mL of PBS to the top of the resin bed and centrifuging at 1,000 × g for 2 minutes. Discard the flowthrough and repeat this step a total of 3 times.
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. Purify the Azide‑labeled protein
5.1 Transfer the equilibrated column into a new collection tube.
5.2 Carefully pipette the entire reaction mixture (2-4mL) onto the center of the column.
5.3 Centrifuge the column‑tube assembly at 1,000 × g for 2 minutes. The purified Azide‑labeled protein is in the collection tube.
ZJ26F0434377
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