生物素长臂抗体标记试剂盒（Biotin-LC-LC-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-LC-LC-NHS试剂、缓冲液及离心式脱盐柱。盒内提供的Biotin-LC-LC-NHS试剂是一种双长链、NHS 酯活化的生物素化试剂，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，NHS-LC-LC-Biotin为脂溶性长臂，增加了生物素基团和蛋白的空间距离，降低空间结合阻碍，对于链霉亲和素的结合可能更有效。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100 μg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。
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图1 微型长臂生物素抗体标记试剂盒(Biotin-LC-LC-NHS) (M1510185)标记原理图
Biotin-LC-LC-NHS特点
•蛋白标记——使抗体或其他蛋白生物素化，以便使用链霉素亲和素探针或树脂进行检测或纯化
•膜可透过性——可用于细胞内部（细胞内）标记
•胺反应性——与伯胺(-NH2)发生反应，如赖氨酸(K)的侧链或多肽的氨基末端
•不可逆——形成永久性酰胺键；间隔臂不能被裂解
•溶解度——必须溶于DMSO或DMF中，然后在水性缓冲液中进一步稀释
•可触及的空间距离较长—间隔臂（添加至靶标的总长度）为30.5埃；由13原子链延长的生物素戊酸基团组成
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. 脱盐柱建议一次性使用。
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. 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；
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，则将其稀释至1mg/mL，并加入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，通过向抗体中加入适量的0.1M NaHCO₃缓冲液来制备1mg/mL的抗体溶液。用超纯水将1M溶液稀释10倍，制备0.1M NaHCO₃缓冲液。
2.2 Biotin-LC-LC-NHS准备：向Biotin-LC-LC-NHS试剂管中加入2 μL DMSO，涡旋使生物素完全溶解；
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 将准备好的100μL抗体溶液加入到生物素试剂管中。盖上管盖，轻轻翻转几次，使生物素试剂完全溶解。
2.4 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 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。
3.2 向脱盐柱树脂顶部加入500μL PBS，轻轻拧上脱盐柱上盖，1000×g离心2分钟，丢弃溶液
3.3 重复步骤3.2两次，第三次洗涤时加入PBS，直到进入下一步骤时再进行离心；
3.4 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；
4. 生物素化抗体纯化
4.1 将准备好的脱盐柱转移至新的收集管中，并将步骤2.4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。
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，可稳定保存半年。或加入等体积甘油于-20°C 存 储，可稳定保存半年。
The NHS-LC-LC-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-LC-LC-NHS, buffer, and purification columns. The Biotin-LC-LC-NHS reagent included in the kit is a dual long-chain, NHS ester-activated biotinylation reagent suitable 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, NHS-LC-LC-Biotin is a lipophilic long-arm biotinylation reagent that increases the spatial distance between the biotin group and the protein, reducing steric hindrance and potentially leading to more efficient binding to streptavidin. 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 100 μg 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 Micro NHS-LC-LC-Biotinylation Kit (M1510185) Labeling Principle Schematic
Features of Biotin-LC-LC-NHS:
• Protein labeling—biotinylate antibodies or other proteins for detection or purification using streptavidin probes or resins
• Membrane Permeable-Suitable for intracellular (inside the cell) labeling.
• Amine-reactive—reacts with primary amines (-NH2), such as the side-chain of lysines (K) or the amino-termini of polypeptides
• Irreversible—forms permanent amide bonds; spacer arm cannot be cleaved
• Solubility-Must be dissolved in DMSO or DMF, followed by further dilution in an aqueous buffer.
• Extended Accessible Spacing-The spacer arm (total length added to the target) is 30.5 Å; composed of a biotin valeric acid group extended by a 13-atom chain.
Required materials not supplied
1. Microcentrifuge capable of 1,000 xg.
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. Concentrate the antibody to ≥1 mg/mL.
4. Do not reuse the purification resin.
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 reactive dyes should be protected from prolonged exposure to light.
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 of ≥1 mg/mL and is in an appropriate buffer, dilute it to 1 mg/mL, and add a 10% volume of 1 M sodium bicarbonate buffer. If the antibody is a powder lyophilized from an appropriate buffer, prepare a 1 mg/mL solution of the antibody by adding an appropriate amount of 0.1 M sodium bicarbonate buffer to the antibody. Prepare 0.1 M sodium bicarbonate buffer by diluting the 1 M solution 10-fold with ultrapure water.
2.2 Biotin-LC-LC-NHS Preparation: Add 2 μL of DMSO to the Biotin-LC-LC-NHS reagent tube and vortex to ensure the biotin is completely dissolved.
Note: To visually ensure that the dye has fully dissolved, peel the label off the vial of biotin reagent.
2.3 Add the prepared 100 μL of the antibody solution to the vial of biotin reagent. Cap the vial and gently invert it a few times to fully dissolve the biotin reagent.
2.4 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 Loosen the cap on a spin column, twist the tab off of the bottom, then place the column into a collection tube. Centrifuge the column‑tube 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.
3.2 Add 500 μL of PBS Buffer, then centrifuge the column‑tube assembly at 1,000 x g for 2 minutes to equilibrate the column.
3.3 Repeat step 3.2 for two additional times. Add PBS for the third wash but wait to centrifuge until immediately prior to proceeding to step.
3.4 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 Insert prepared desalting column into the new Collection Tube and apply the entire sample from step 2.4 to the column.
4.2 Centrifuge the column‑tube assembly at 1,000 × 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 six months. Alternatively, add an equal volume of glycerol and store at -20°C for stable preservation up to six months.
ZJ26F0434360
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