生物素抗体标记试剂盒（Biotin-PEG4-NHS）专为高效可靠的抗体标记而设计，仅需60分钟即可获得即用型标记抗体，且操作时间极短。试剂盒包含抗体标记所需的所有必要试剂，包括Biotin-PEG4-NHS、缓冲液、纯化填料和离心柱管。盒内提供的Biotin-PEG4-NHS试剂是一种聚乙二醇化、水溶性的生物素化试剂，适用于标记含有伯胺基的抗体、蛋白质及其他分子。NHS活化生物素可在pH 7–9的缓冲液中与伯氨基（-NH2）高效反应，形成稳定的酰胺键（图1）。同时，聚乙二醇使生物素具有水溶性，可以有效防止生物素化抗体聚集。试剂盒中的离心式脱盐柱用于从过量生物素试剂中纯化生物素化抗体，回收率达70–95%。本试剂盒所含试剂足以完成10次、每次1 mg抗体的生物素化反应。生物素化抗体可通过亲和素或链霉亲和素探针，应用于ELISA、Western Blot、免疫荧光/免疫细胞化学（IF/ICC）或流式细胞术等检测。Aladdin提供一系列高性能链霉亲和素偶联物，用于检测生物素化抗体的性能。
图1 生物素抗体标记试剂盒(Biotin-PEG4-NHS) (N1510179)标记原理图
Biotin-PEG4-NHS特点
• 蛋白质标记——可对抗体或其他蛋白质进行生物素化，便于通过链霉亲和素树脂或探针进行固定、纯化或检测
• 胺基反应性——可与伯氨基（-NH2）（如赖氨酸侧链或多肽氨基末端）发生反应
• 聚乙二醇化——间隔臂含亲水性的四单元聚乙二醇 (PEG) 基团
• 提高溶解度——聚乙二醇化赋予生物素化分子水溶性，有助于防止溶液中储存的生物素化抗体发生聚集
• 不可逆结合——形成永久性酰胺键；间隔臂不可断裂
• 长臂结构——间隔臂（添加至目标分子的总长度）为29埃；这可减少与亲和素分子结合时的空间位阻
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）。
重要须知
1. 纯化的抗体缓冲液中不得含有铵离子或者伯胺组分，例如Tris、甘氨酸、乙醇胺、三乙胺、谷胱甘肽或咪唑，所有这些物质都会显著抑制蛋白质标记；
2. 用于标记的抗体需经过纯化，不含有BSA或明胶等稳定剂；
3. 抗体浓度1-2mg/mL最佳，浓度低于1mg/mL, 标记效率可能会降低，浓度过高，抗体体积会过少，不利于纯化回收；
4. 待标记抗体体积应控制在0.4-0.7mL, 保证纯化柱的最佳去除效果；
5.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；
6. 本试剂盒中的纯化填料建议一次性使用；
7. 操作过程中，务必穿戴实验服、一次性手套及防护装备；
8. 所有产品仅限于研究用途。
使用说明
1. 缓冲液配制：
1.1 1M NaHCO₃的配制。根据样品量配制适量的1M NaHCO₃溶液，如称取42mg NaHCO₃，加入0.5mL超纯水即得1M NaHCO₃溶液。NaHCO₃可维持标记反应体系的pH在7-9之间，提高标记效率。注：NaHCO₃溶液须现用现配。
2. 抗体标记
2.1 如果待标记的抗体浓度≥1mg/mL，并且在适当的缓冲液中，加入10%抗体体积的1M NaHCO₃缓冲液。如果抗体是从适当的缓冲液中冻干的粉末，则将1 mg抗体溶解于0.4-0.7mL 0.1M NaHCO₃缓冲液中。用超纯水将1M溶液稀释10倍，制备0.1M NaHCO₃缓冲液。
2.2 将准备好的抗体溶液转移到生物素试剂管中。盖上管盖，轻轻翻转几次，使生物素试剂完全溶解。
注意：为确保染料完全溶解，请将标签从生物素试剂管上撕下。
2.3 将反应管室温避光反应1h。反应可以在振荡器或混合器上进行，建议上下翻转速度为25rpm。如果在反应过程中不使用混合仪器，则应每10分钟将反应溶液倒置混合一次。
注意：反应进行期间，可提前进行步骤3的操作，完成脱盐柱的平衡。
3. 脱盐柱预平衡
3.1 将一个空的离心柱放入一个 15mL 收集管中。
3.2 通过反复倒置试剂瓶重悬纯化树脂。然后取3.0 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)。
3.3 向脱盐柱树脂顶部加入2mL PBS，1000×g离心2分钟，弃去流出液；
3.4 重复步骤3.3两次，第三次洗涤时加入PBS，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。
3.5 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；
4. 生物素化抗体纯化
4.1 将准备好的脱盐柱放入新的15 mL离心管中，并将步骤2.2中的全部样品加入脱盐柱中；
4.2 脱盐柱1000xg离心2分钟，收集管中的流出即为所需收集的生物素化抗体。
5. 测定蛋白浓度（可选）
5.1 通过检测280nm吸光度检测抗体浓度。
6. 保存
6.1 向标记后的蛋白中加入 0.05~0.2 % Proclin 300 或 0.05 %叠氮化钠，及蛋白稳定剂 (如 0.1 % BSA)，于 2~8°C 避光保存，可稳定保存数月。如需长期储存，请将溶液分装成单次使用的小份，于 -20°C 冷冻。避免反复冻融。
The NHS-PEG4-Biotinylation Kit is designed for efficiency and reliability, delivering ready-to-use labeled antibody in just 60 minutes, with minimal hands-on time. The kit include all necessary reagents for antibody labeling, including Biotin-PEG4-NHS, buffers, purification resin, and empty spin columns. The Biotin-PEG4-NHS reagent included in the kit is a pegylated, water-soluble reagent for labeling antibodies, proteins, and other molecules that have primary amines. NHS-activated biotins react efficiently with primary amino groups (-NH2) in pH 7-9 buffers to form stable amide bonds (Fig 1). Meanwhile, pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution. Spin columns included in the kit are used for purifying the biotinylated antibody from excess biotin reagent with yields of 70–95%. The kit contains sufficient reagents for 10 biotinylation reactions of 1 mg of antibody. The biotinylated antibody can be detected in ELISA, Western Blot, IF/ICC or Flow Cytometry application using avidin or streptavidin probes. Aladdin offers a series of high-performance streptavidin conjugates designed for detecting the performance of biotinylated antibodies.
Fig. 1 NHS-PEG4-Biotinylation Kit (N1510179) Labeling Principle Schematic
Features of Biotin-PEG4-NHS:
• Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins
• Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
• Pegylated – spacer arm contains a hydrophilic, 4-unit, polyethylene glycol (PEG) group
• Enhances solubility—pegylation imparts water solubility to the biotinylated molecule, helping to prevent aggregation of biotinylated antibodies stored in solution
• Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved
• Long reach—spacer arm (total length added to target) is 29 angstroms; this reduces steric hindrance when binding to avidin molecules
Required materials not supplied
1. Microcentrifuge capable of 1,000 x g.
2. Desired antibody for labeling (free of BSA or any carrier protein).
3. PBS buffer (pH 7.2-7.4).
Important
1. The purified antibody should be in a buffer that does not contain primary amines (for example, ammonium ions, Tris, glycine, ethanolamine, triethylamine, glutathione) or imidazole. All of these substances significantly inhibit protein labeling.
2. Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.
3. An antibody concentration of 1–2 mg/mL is optimal. Concentrations below 1 mg/mL may reduce labeling efficiency, while excessively high concentrations may result in too small a volume of antibody, which is unfavorable for purification recovery.
4. The volume of the antibody to be labeled should be controlled between 0.4 mL and 0.7 mL to ensure optimal removal efficiency using the purification column.
5. 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.
6. The purification resin included in this kit is recommended for single use only.
7. During operation, always wear a lab coat, disposable gloves, and protective equipment.
8. All products are for research use only.
Instruction for use
1. Prepare the reagents
1.1 Prepare a 1 M sodium bicarbonate solution—Prepare an appropriate amount of 1M NaHCO₃ solution based on the sample volume. For example, weigh 42mg of NaHCO₃ and add 0.5mL of ultrapure water to obtain a 1M NaHCO₃ solution. NaHCO₃ can maintain the pH of the labeling reaction system between 7-9, thereby improving labeling efficiency. Note: The NaHCO₃ solution must be prepared fresh before each use.
2. Label the antibody
2.1 If the antibody to be labeled has a concentration ≥1 mg/mL and is in an appropriate buffer, add 10% of the antibody volume of 1 M NaHCO₃. If the antibody is a lyophilized powder from an appropriate buffer, dissolve 1 mg of the antibody in 0.4–0.7 mL of 0.1 M NaHCO₃. Prepare 0.1 M NaHCO₃ buffer by diluting the 1 M solution 10-fold with ultrapure water.
2.2 Transfer prepared antibody solution to the vial of biotin reagent. Cap the vial and gently invert it a few times to fully dissolve the biotin reagent.
Note: To visually ensure that the dye has fully dissolved, peel the label off the vial of biotin reagent.
2.3 Incubate the solution for 60 minutes at room temperature. The reaction can be carried out on a shaker or mixer, recommended speed for flipping up and down is 25 rpm. If a mixing instrument is not used during the reaction process, the reaction solution should be mixed upside down every 10 minutes.
Note: During the incubation period, proceed to steps 3 below, to prepare a spin column for the purification of the labeled protein.
3. Prepare the spin column
3.1 Place an empty spin column in a 15 mL collection tube.
3.2 Suspend the purification resin by repeated inversion of the reagent bottle. Then add 3.0 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).
3.3 Add 2 mL of PBS Buffer, then centrifuge the column-tube assembly at 1000 x g for 2 minutes to equilibrate the column.
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.
3.4 Repeat step 3.3 for two additional times. Add PBS for the third wash but wait to centrifuge until immediately prior to proceeding to step.
3.5 After the third wash check there is no solution in the column, if required briefly centrifuge until no solution remains.
4. Purification with Desalting Column
4.1 Place column into a new 15mL collection tube and apply the entire sample from step 2.2 to the column.
4.2 Centrifuge the column‑tube assembly at 1000 × g for 2 minutes. The purified antibody conjugate is in the collection tube.
5. Determine the antibody concentration (Optional)
5.1 Determination of antibody concentration by measuring absorption at 280 nm.
6. Strorage
6.1 Add 0.05–0.2% Proclin 300 or 0.05% sodium azide, along with a protein stabilizer (such as 0.1% BSA), to the labeled protein. Store protected from light at 2–8°C for stable preservation up to several months. For long-term storage, divide the solution into single-use aliquots and freeze at -20°C. Avoid repeated freeze-thaw cycles
H2621669
危险品化学品经营许可证（带存储）