AF荧光染料叠氮化物通过铜催化的点击反应（CuAAC）与炔烃反应。这种点击化学在多种生物学应用中的使用越来越广泛。AF染料叠氮化物可通过铜催化的点击反应与末端炔烃发生反应。除了作为与流式细胞分析、显微镜和 HCS 配合使用的极为明亮且光稳定的荧光基团外，还可作为生物正交或生物学独特的半抗原用于需要信号放大的应用。
荧光叠氮染料标记炔基修饰的生物分子方案
一、荧光叠氮染料标记寡核苷酸
1. 准备以下储备溶液：
200 mM THPTA（三（3-羟丙基三唑基甲基）胺）水溶液
100 mM CuSO₄水溶液
炔基修饰的寡核苷酸水溶液（尽可能浓缩，例如>10 mg/mL）
100 mM抗坏血酸钠水溶液
10 mM荧光叠氮染料在DMSO或水中。
2. 将CuSO₄与THPTA以1:2的比例混合并涡旋几分钟，该工作溶液冷冻后可稳定保存数周。
3. 向炔基修饰的寡核苷酸溶液中加入过量的荧光叠氮染料（摩尔比为2-5eq.）。
4. 加入5eq.的THPTA/CuSO₄工作溶液（来自步骤1）。
5. 加入10-30eq.的抗坏血酸钠。
6. 在室温下搅拌、涡旋或摇动反应混合物30-60分钟。
7. 用乙醇沉淀寡核苷酸或通过您选择的方法（如HPLC）进行纯化。
二、荧光叠氮染料标记肽
1. 准备以下储备溶液：
200 mM THPTA配体水溶液
100 mM CuSO₄水溶液
炔基修饰的肽在水或DMF中（根据肽的溶解度，尽可能浓缩至>10 mg/mL）
100 mM抗坏血酸钠水溶液
10 mM荧光叠氮染料在DMSO或水中。
2. 将CuSO₄与THPTA配体以1:2的比例孵育几分钟，该溶液冷冻后可稳定保存数周。
3. 向炔基修饰的肽溶液中加入过量的荧光叠氮染料（摩尔比为5-10eq.）。
4. 加入5-10eq.的THPTA/CuSO₄。
5. 加入10-20eq.的抗坏血酸钠。
6. 在室温下搅拌、涡旋或摇动反应混合物30-60分钟。
7. 通过HPLC纯化所需的肽。
三、荧光叠氮染料标记小分子有机炔基化合物
1. 准备以下储备溶液：
200 mM THPTA配体水溶液
100 mM CuSO₄水溶液
炔基化合物在水或DMF中（根据化合物的溶解度，尽可能浓缩至>10 mg/mL）
100 mM抗坏血酸钠水溶液
10 mM荧光叠氮染料在DMSO或水中
2. 将CuSO₄与THPTA配体以1:2的比例孵育几分钟，该溶液冷冻后可稳定保存数周。
3. 向炔基溶液中加入过量的荧光叠氮染料（摩尔比为5-10eq.）。
4. 加入25eq.的THPTA/CuSO₄。
5. 加入50eq.的抗坏血酸钠。
6. 在室温下搅拌反应混合物30-60分钟。
7. 通过色谱法或其他方法纯化所需的分子。
四、荧光叠氮染料标记生物聚合物
1. 准备以下储备溶液：
200 mM THPTA配体水溶液
100 mM CuSO₄水溶液
炔基修饰的生物聚合物水溶液（尽可能浓缩，例如>5 mg/mL）
100 mM抗坏血酸钠水溶液
10 mM荧光叠氮染料在DMSO或水中
2. 将CuSO₄与THPTA配体以1:2的比例孵育几分钟，该溶液冷冻后可稳定保存数周。
3. 向炔基修饰的生物聚合物溶液中加入过量的荧光叠氮染料（负载比：5-20荧光叠氮染料/炔基）。
4. 加入与荧光叠氮染料摩尔量相当的5eq.THPTA/CuSO₄。
5. 加入与荧光叠氮染料摩尔量相当的10eq.抗坏血酸钠。
6. 在室温下搅拌、涡旋或摇动反应混合物30-60分钟。
7. 通过凝胶过滤或透析纯化所需的分子。
五、荧光叠氮染料标记细胞、细胞裂解物或生物样本
1. 准备以下点击反应溶液：
100 mM THPTA配体在水缓冲液或水中
20 mM CuSO₄水溶液
300 mM抗坏血酸钠水溶液
2.5 mM炔基或叠氮标记试剂在水或DMSO中
2. 对于每个叠氮或炔基修饰的细胞或细胞裂解物样本，向1.5 mL微量离心管中加入以下试剂，然后短暂涡旋混合：50μl 细胞或细胞裂解物样本50μl PBS缓冲液50μl 5 mM对应的荧光叠氮染料（或染料炔基）检测试剂在DMSO或水中
3. 加入10μl 100 mM THPTA溶液，短暂涡旋混合。
4. 加入10μl 20 mM CuSO₄溶液，短暂涡旋混合。
5. 加入10μl 300 mM抗坏血酸钠溶液以启动点击反应，短暂涡旋混合。
6. 避光保护反应，并在室温下孵育30分钟。
7. 细胞或细胞裂解物现已完成点击标记，准备进行下游处理和分析。
Azide derivatives of AF fluorescent dyes react with alkynes via copper-catalyzed azide-alkyne cycloaddition (CuAAC). This click chemistry is increasingly used in a variety of biological applications. AF dye azides share the same chemical structure as AF dye azides and can react with terminal alkynes through CuAAC. Besides serving as extremely bright and photostable fluorophores for use with flow cytometry, microscopy, and HCS (High Content Screening), they also function as bioorthogonal or biologically unique haptens in applications requiring signal amplification.
Protocol for Labeling Alkyne-Modified Biomolecules with Fluorescent Azide Dyes
I. Labeling Oligonucleotides with Fluorescent Azide Dyes
Prepare the following stock solutions:
- 200 mM THPTA (Tris(3-hydroxypropyltriazolylmethyl)amine) aqueous solution
- 100 mM CuSO₄ aqueous solution
- Alkyne-modified oligonucleotide aqueous solution (as concentrated as possible, e.g., >10 mg/mL)
- 100 mM sodium ascorbate aqueous solution
- 10 mM fluorescent azide dye in DMSO or water.
- Mix CuSO₄ and THPTA at a 1:2 ratio, vortex for several minutes. The working solution is stable for weeks when frozen.
- Add an excess of fluorescent azide dye to the alkyne-modified oligonucleotide solution (molar ratio: 2–5 eq.).
- Add 5 eq. of the THPTA/CuSO₄ working solution (from Step 2).
- Add 10–30 eq. of sodium ascorbate.
- Stir, vortex, or shake the reaction mixture at room temperature for 30–60 minutes.
- Purify the oligonucleotide by ethanol precipitation or your method of choice (e.g., HPLC).
II. Labeling Peptides with Fluorescent Azide Dyes
Prepare the following stock solutions:
- 200 mM THPTA ligand aqueous solution
- 100 mM CuSO₄ aqueous solution
- Alkyne-modified peptide in water or DMF (as concentrated as possible to >10 mg/mL, depending on peptide solubility)
- 100 mM sodium ascorbate aqueous solution
- 10 mM fluorescent azide dye in DMSO or water.
- Incubate CuSO₄ and THPTA ligand at a 1:2 ratio for several minutes. The solution is stable for weeks when frozen.
- Add an excess of fluorescent azide dye to the alkyne-modified peptide solution (molar ratio: 5–10 eq.).
- Add 5–10 eq. of THPTA/CuSO₄.
- Add 10–20 eq. of sodium ascorbate.
- Stir, vortex, or shake the reaction mixture at room temperature for 30–60 minutes.
- Purify the desired peptide by HPLC.
III. Labeling Small-Molecule Organic Alkyne Compounds with Fluorescent Azide Dyes
Prepare the following stock solutions:
- 200 mM THPTA ligand aqueous solution
- 100 mM CuSO₄ aqueous solution
- Alkyne compound in water or DMF (as concentrated as possible to >10 mg/mL, depending on compound solubility)
- 100 mM sodium ascorbate aqueous solution
- 10 mM fluorescent azide dye in DMSO or water.
- Incubate CuSO₄ and THPTA ligand at a 1:2 ratio for several minutes. The solution is stable for weeks when frozen.
- Add an excess of fluorescent azide dye to the alkyne solution (molar ratio: 5–10 eq.).
- Add 25 eq. of THPTA/CuSO₄.
- Add 50 eq. of sodium ascorbate.
- Stir the reaction mixture at room temperature for 30–60 minutes.
- Purify the desired molecule by chromatography or other methods.
IV. Labeling Biopolymers with Fluorescent Azide Dyes
Prepare the following stock solutions:
- 200 mM THPTA ligand aqueous solution
- 100 mM CuSO₄ aqueous solution
- Alkyne-modified biopolymer aqueous solution (as concentrated as possible, e.g., >5 mg/mL)
- 100 mM sodium ascorbate aqueous solution
- 10 mM fluorescent azide dye in DMSO or water.
- Incubate CuSO₄ and THPTA ligand at a 1:2 ratio for several minutes. The solution is stable for weeks when frozen.
- Add an excess of fluorescent azide dye to the alkyne-modified biopolymer solution (loading ratio: 5–20 fluorescent azide dyes per alkyne).
- Add 5 eq. of THPTA/CuSO₄ relative to the molar amount of the fluorescent azide dye.
- Add 10 eq. of sodium ascorbate relative to the molar amount of the fluorescent azide dye.
- Stir, vortex, or shake the reaction mixture at room temperature for 30–60 minutes.
- Purify the desired molecule by gel filtration or dialysis.
V. Labeling Cells, Cell Lysates, or Biological Samples with Fluorescent Azide Dyes
Prepare the following click reaction solutions:
- 100 mM THPTA ligand in aqueous buffer or water
- 20 mM CuSO₄ aqueous solution
- 300 mM sodium ascorbate aqueous solution
- 2.5 mM alkyne or azide labeling reagent in water or DMSO.
- For each azide- or alkyne-modified cell or cell lysate sample, add the following reagents to a 1.5 mL microcentrifuge tube and briefly vortex to mix: 50 μL cell or cell lysate sample, 50 μL PBS buffer, 50 μL 5 mM corresponding fluorescent azide dye (or dye alkyne) detection reagent in DMSO or water.
- Add 10 μL of 100 mM THPTA solution and briefly vortex to mix.
- Add 10 μL of 20 mM CuSO₄ solution and briefly vortex to mix.
- Add 10 μL of 300 mM sodium ascorbate solution to initiate the click reaction, then briefly vortex to mix.
- Protect the reaction from light and incubate at room temperature for 30 minutes.
- The cells or cell lysates are now click-labeled and ready for downstream processing and analysis.
危险品化学品经营许可证（带存储）