Cy7.5 maleimide, a CY dye (short for Cyanine), consists of two nitrogen atoms connected by an odd number of methyl units. Cyanine compounds are characterized by long wavelength, adjustable absorption and emission, high extinction coefficient, good water solubility, and relatively simple synthesis [1]. CY dyes are often used for labeling proteins, antibodies, and small molecular compounds. For labeling 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 [2].

蛋白 (抗体) 原液准备: 蛋白浓度应配制为 2 mg/mL，pH 值为 8.5±0.5，低于 8.0 时用 1 M 碳酸氢钠调整。若蛋白浓度低于 2 mg/mL，建议最终浓度为 2-10 mg/mL。蛋白不应含伯胺和铵离子。染料原液准备: 将无水 DMSO 稀释CY染料制成 10 mM 储备溶液，存于 -20 ℃ 或 -80 ℃ 避光保存。染料工作液用量计算: CY染料与蛋白最佳摩尔比为 10。例: 标记 500 μL 2 mg/mL IgG (MW=150,000) 用 100 μL DMSO 溶解1 mg CY 染料，所需 CY 体积为 3.95 μL。使用方法1. 标记反应: 将 10 mg/mL CY 染料缓慢加入 0.5 mL 蛋白样品溶液中，轻轻摇匀混合，短暂离心，室温避光轻轻摇晃孵育 60 分钟，每 10–15 分钟轻轻颠倒。2. 蛋白纯化脱盐：使用 SepHadex G-25 柱按生产说明书纯化。将反应混合物装入柱内，加 PBS (pH 7.2-7.4) 完成纯化。结合含有所需的染料-蛋白质缀合物的组分。