磺化Cy7-羧酸sulfo-cy7 carboxylic acid

CY7 is a CY dye. CY, short for Cyanine, is a compound consisting of two nitrogen atoms connected by an odd number of methyl units. Cyanine compounds have the characteristics of long wavelength, adjustable absorption and emission, high extinction coefficient, good water solubility and relatively simple synthesis. CY dyes are of en used for the labeling of proteins, antibodies and small molecular compounds. For the labeling of protein antibodies, the combination can be completed through a simple mixing reaction. Below, we introduce the labeling method of protein antibody labeling, which has certain reference significance.

Solid

原液制备
1. 蛋白准备
为了获得最佳标记效果，请将蛋白 (抗体) 浓度配制为 2 mg/mL。
1) 蛋白溶液 pH 值应为 8.5±0.5，若 pH 低于 8.0，则用 1 M 碳酸氢钠进行调整。
2) 若蛋白浓度低于 2 mg/mL，标记效率会大大降低，为了获得最佳标记效率，建议最终蛋白质浓度范围为 2-10 mg/mL。
3) 蛋白必须在不含伯胺 (如 Tris 或甘氨酸) 和铵离子的缓冲液中，否则会影响标记效率。
2. 染料准备
将无水 DMSO 稀释 CY 染料，制成 10 mM 储备溶液。通过玻璃管或旋涡充分混合。
注：CY 储存液建议分装后于 -20 ℃ 或 -80 ℃ 避光保存。
3. 染料工作液用量计算
标记反应所需的 CY 染料用量取决于要标记蛋白的用量，CY 染料与蛋白的最佳摩尔比为 10 左右。
例：假如所需标记蛋白为 500 μL 2 mg/mL 的 IgG (MW=150,000),用 100 μL DMSO 溶解一管 1 mg CY 染料，则所需 CY 体积为 3.95 μL，详细计算流程如下 (以 CY3-NHS ester 为例) ：
1) mmol (IgG) = mg/mL (IgG) ×mL (IgG) / MW (IgG) =2 mg/mL×0.5 mL / 150,000 mg/mmol= 6.7×10-6 mmol
2) mmol (CY3-NHS ester) = mmol (IgG) ×10 =6.7×10-6 mmol×10 = 6.7×10-5 mmol
3) μL (CY3-NHS ester) = mmol (CY3-NHS ester) ×MW (CY3-NHS ester) / mg/μL (CY3-NHS ester) = 6.7×10-5 mmol× 590.15 mg/mmol / 0.01 mg/μL =3.95 μL (CY3-NHS ester)


使用方法
1. 标记反应
1) 取算好体积的新鲜配制的 10 mg/mL CY 染料缓慢加入到 0.5 mL 蛋白样品溶液中，轻轻摇匀混合，然后短暂离心将样品收集在反应管底部。切忌剧烈混匀，防止蛋白样品变性失活。
2) 将该反应小管置于避光处，在室温条件下轻轻摇晃孵育 60 分钟，每隔 10–15 分钟，将反应小管轻轻颠倒几次，以充分混合两种反应物，提高标记效率。
2. 蛋白纯化脱盐
以下方案是使用 SepHadex G-25 柱纯化染料蛋白偶联物为例。
1) 按照生产说明书制备 SepHadex G-25 柱。
2) 将反应混合物装入 SepHadex G-25 色谱柱顶部。
3) 当样品运行到顶部树脂表面下方时，立即加入 PBS (pH 7.2-7.4)。
4) 向所需样品中加入更多的 PBS (pH 7.2-7.4),完成柱纯化。结合含有所需要的染料-蛋白质缀合物的组分。

Medlife has not independently confirmed the accuracy of these methods. They are for reference only.

Room temperature or refrigerated transportation.

4°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

• Description: CY7 labels proteins, antibodies, peptides, and oligonucleotides with red fluorescence, it can be used in vivo imaging system.
  Method: For detecting biodistribution of CY7-labeled ASP nanocomposite.
  1. Testing mice are intravascularly injected with CY7-labeled ASP nanocomposite.
  2. The imaging is performed at 3, 6, 12, 24, and 48 h post-injection using IVIS Spectrum Imaging System. After imaging, mice are sacrificed and major organs (heart, liver, spleen, lung, and kidney) and tumors are harvested for ex vivo imaging.
  3. The samples are measured in triplicate to ensure reproducibility and accuracy. Fluorescence signals are quantified by Spectrum Living Image Software.
• Description: CY7 labels proteins, antibodies, peptides, and oligonucleotides with red fluorescence, it can be used in vivo imaging system.
  Method: For detecting biodistribution of CY7-labeled polydopamine nanoparticles.
  1. CY7-labeled polydopamine nanoparticles (50 μL; 100 μg/mL) is injected intraarticularly into the left temporomandibular joint osteoarthritis of SD rats.
  2. In vivo imaging system (Lumina Series III, PerkinElmer, USA) imaging is documented instantly and at 1, 3, 5, 7 days after the injection to evaluate the distribution and retention profile of nanoparticles.
• Description: CY7 labels proteins, antibodies, peptides, and oligonucleotides with red fluorescence, it can be used in vivo imaging system.
  Method: For CY7-labeled nanoparticles cellular uptake assay.
  1. Cells are seeded in a 24-well plate at a density of 3×10 cells per well and incubated with 5 μg/mL of Cy7-labeled nanoparticles as a control.
  2. After incubation for 6 h, the nuclei, cytoskeleton, and AM or AMPG are stained with Cy7 (40 min).
  3. Use a confocal laser scanning microscopy for image.

• [1]. Ptaszek M. Rational design of fluorophores for in vivo applications. Prog Mol Biol Transl Sci. 2013;113:59-108.  [Information]

  [2]. Lim B, et al. A Unique Recombinant Fluoroprobe Targeting Activated Platelets Allows In Vivo Detection of Arterial Thrombosis and Pulmonary Embolism Using a Novel Three-Dimensional Fluorescence Emission Computed Tomography (FLECT) Technology. Theranostics. 2017 Feb 26;7(5):1047-1061.  [Information]

  [3]. Shindy, H. A. (2017). Fundamentals in the chemistry of cyanine dyes: A review. Dyes and Pigments, 145, 505–513. doi:10.1016/j.dyepig.2017.06.029

产品不同，其溶解度不同。建议根据产品选择合适的溶剂配制储备溶液；配成溶液后，建议分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (protect from light)。-80°C 储存时，建议在 6 个月内使用，-20°C 储存时，建议在 1 个月内使用。