BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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阿拉丁荧光染料抗体标记试剂盒设计注重效率与可靠性，可在90分钟内快速完成标记，仅需极少的人工操作时间。试剂盒内含抗体标记所需的所有试剂，包括NHS酯活化染料、缓冲液和脱盐柱。试剂盒中的活性染料可与抗体或蛋白质中的赖氨酸残基高效结合，生成共价连接的荧光传感器。配套的脱盐柱可去除过量未反应的染料，回收率可达70%-95%。试剂盒提供5管活性染料，每管活性染料都含有优化量的染料，可实现1mg抗体的标记反应。
我们提供多种颜色染料试剂盒，包可兼容多种检测系统，包括荧光显微镜和流式细胞术。
对于标记少量抗体（~100 μg），我们推荐使用荧光染料抗体标记试剂盒（100 μg）。
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. 抗体浓度最佳浓度2mg/mL，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. 离心脱盐柱的样品体积必须在400至700 μL之间，以确保最佳性能。超出此范围的样品体积可能导致抗体回收率降低或未偶联试剂去除不彻底。
5. 脱盐柱建议一次性使用。
6. 试剂盒使用之前，需将所有组分恢复至室温。
7. 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。
使用说明
1. 缓冲液配制：
a) 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
a) 如果待标记的抗体浓度≥2mg/mL，并且在适当的缓冲液中，则将其稀释至2mg/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2mg/mL的抗体溶液。用超纯水将1M NaHCO₃缓冲液稀释10倍，即得到0.1M NaHCO₃缓冲液。
b) 取一管活性染料，加入10μL DMSO，涡旋至染料溶解。
注意：为确保染料完全溶解，请将标签从活性染料管上撕下。
c) 将0.5mL抗体溶液转移到活性染料管中。盖上盖子，轻轻翻转几次，使染料和抗体混匀。
d) 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
a) 取离心柱管放在离心管架上，打开盖子，取下管底的红色堵头。
b) 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，树脂自然沉降，柱内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 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)。
c) 通过在树脂床顶部加入 2mL PBS，以 1,000 × g 离心 2 分钟，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。
4. 荧光标记抗体纯化
a) 小心的将反应管中的反应液加入脱盐柱中，盖上脱盐柱盖子，注意盖子轻轻拧上即可。
b) 脱盐柱1000 × g离心2分钟，收集管中的流出即为所需收集的抗体偶联物。
5. 测定抗体浓度和DOL（可选）
DOL（标记度）是衡量荧光标记效率的关键参数，表示荧光团与目标分子（如抗体或蛋白质）的摩尔比。可以从抗体偶联物的吸收光谱中测定。
a) 检测标记产物280nm的吸光度（A₂₈₀）和对应染料最大吸收波长处的吸光度（Adye）。不同染料的参数可参考下面表格。
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b) 计算抗体偶联物中抗体浓度：
抗体浓度（mg/mL） =(A280 - CF280 × Adye) / 1.4 c)
计算DOL:
DOL = (Adye / εdye) / [(A280 - CF280 × A280) / 210,000]
注意：
1. 典型的IgG抗体在280nm处的摩尔消光系数为210,000M-1cm-1。
2. Adye 是指染料在最大吸收波长处的吸光度值。
3. CF280 指染料在280nm处的校正系数。
6. 保存
a) 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 Aladdin’s Fluorescent Dye Antibody Labeling Kits are designed for efficiency and reliability, delivering ready-to-use labeled proteins in just 90 minutes, with minimal hands-on time. The kit contains all reagents required for antibody labeling, including amine-reactive dye, buffer, and purification columns. The reactive dyes contained in these kits readily react with lysine residues in antibodies or proteins to yield a covalently attached fluorescence-based sensor. Spin columns included in the kits are used for purifying the labeled antibody from excess dye with yields of 70–95%. The kit provides 5 vials of reactive dye, each containing an optimized amount of dye for labeling 1 mg of antibody.
We offer kits with a variety of dye colors, compatible with multiple detection systems, including fluorescence microscopy and flow cytometry.
For labeling smaller amounts of antibodies (∼100 μg), we recommend our Fluorescent Dye antibody labeling kits (100μg).
Required materials not supplied
1. Centrifuge
2. Antibody for labeling (without BSA or any carrier protein)
3. PBS buffer (pH 7.2–7.4)
Important Notes
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. Optimal antibody concentration is 2 mg/mL. Concentrations below 1 mg/mL may reduce labeling efficiency; excessive concentration results in a very small antibody volume, which is unfavorable for purification recovery.
4. The sample volume for the centrifugal desalting column must be between 400 and 700 μL to ensure optimal performance. Volumes outside this range may lead to reduced antibody recovery or incomplete removal of unconjugated reagent.
5. Desalting columns are recommended for single use only.
6. Before using the kit, allow all components to equilibrate to room temperature.
8. All fluorescent dyes are prone to quenching. Please protect from light to minimize fluorescence quenching.
Instructions for Use
1. Prepare the buffer
a) 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
a) If the antibody concentration is ≥2 mg/mL in a suitable buffer, dilute it to 2 mg/mL and add 10% of the antibody volume of 1 M NaHCO₃ buffer. If the antibody is a lyophilized powder from an appropriate buffer, prepare a 2 mg/mL antibody solution by adding an appropriate volume of 0.1 M NaHCO₃ buffer. To prepare 0.1 M NaHCO₃ buffer, dilute the 1 M NaHCO₃ buffer 10-fold with ultrapure water.
b) Take one vial of reactive dye, add 10 μL DMSO, and vortex until the dye is dissolved.
Note: To visually ensure that the dye has fully dissolved, peel the label off the vial of reactive dye.
c) Transfer 0.5 mL of the antibody solution into the reactive dye vial. Cap the vial and gently invert several times to mix the dye and antibody.
d) Incubate the solution for 60 minutes at room temperature in the dark. The reaction can be carried out on a shaker or mixer, recommended speed for flipping up and down is 25rpm. 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 antibody.
3. Prepare the spin column
a) Place the Empty Spin Column on a column rack, open the cap, and remove the red bottom cap.
b) Resuspend the purification resin by inverting the bottle repeatedly. Then take 3.0 mL of the suspension and add it to the column. Allow the resin to settle naturally; the buffer in the column will drain by gravity. Initially, slight pressure may be needed to initiate flow. Centrifuge the column at 1,000 × g for 2 minutes, discard the storage buffer, and place the column back into the same collection tube.
Note: The purification resin is supplied as a 20% ethanol suspension, with a resin-to-ethanol volume ratio of 3:1 (v/v).
c) Add 2 mL of PBS to the top of the resin bed, centrifuge at 1,000 × g for 2 minutes, and discard the flow‑through. Repeat this step a total of three times. The column is now equilibrated.
Note: When using a fixed‑angle rotor, mark the side of the column opposite to the rotor center. For all subsequent centrifugation steps, place the column into the centrifuge with the marked side facing away from the rotor center.
4. Purify the labeled antibody
a) Carefully transfer the reaction solution from the vial onto the column. Cap the column lightly (do not overtighten).
b) Centrifuge the column at 1,000 × g for 2 minutes. The flow‑through collected in the tube is the desired antibody conjugate.
5. Determine the Degree of Labeling (Optional)
The degree of labeling (DOL) is a useful parameter for characterizing the average number of label molecules that have covalently bonded to your sample protein during the labeling reaction. It can be determined from the absorption spectrum of your labeled bioconjugate.
a) Detect the absorbance of the antibody conjugate at 280 nm (A₂₈₀) and the absorbance maximum (λmax) for the respective dye (Adye). The λmax values for commonly used fluorophores are given in the table below.
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Note: Target DOL is the acceptable degrees of labeling (DOL) for a whole IgG.
b) The following formula can be used to calculate the antibody concentration:
Antibody concentration (mg/mL) =(A280 - CF280 × Adye) / 1.4 c)
c) The following formula can be used to calculate the degree of labeling:
DOL = (Adye / εdye) / [(A280 - CF280 × A280) / 210,000]
Note:
1. 210,000 is the molar extinction coefficient (Ec) in M-1cm-1 of IgG at 280nm.
2. Adye is the absorbance at maximum (λmax) for the respective dye.
3. CF280 is the correction factor for the effect of the fluorophore on absorbance at 280nm.
6. Storage
a) 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.
| A1491957 | 组件 | 物理外观 | 5 Reactions | 储存 | 单次测试使用量 |
| A1491957A | AF405 NHS Ester | 类白色固体 | 5 vials | -20℃，避光 | 1瓶可标记1mg抗体 |
| A1491957B | DMSO | 无色透明液体 | 0.5 mL | 室温 | 请按说明配制 |
| A1491957C | 碳酸氢钠 | 白色粉末 | 500 mg | 室温 | 请按说明配制 |
| A1491957D | 纯化填料 | 白色微珠混悬液 | 15 mL | 2-8℃， 禁止冷冻 | 每次反应需使用3mL |
| A1491957E | 离心柱管 | 透明管体 | 5 EA | 室温 | 每次反应需使用1套 |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0737047 |
分析证书 | A1491957 |
| ZJ26F0737046 |
分析证书 | A1491957 |
| ZJ26F0737045 |
分析证书 | A1491957 |
| ZJ26F0737044 |
分析证书 | A1491957 |
| ZJ26F0737043 |
分析证书 | A1491957 |
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