小麦胚芽凝集素 (WGA) 是一种与 N-乙酰基-D-葡萄糖胺和唾液酸结合的凝集素。它是生物应用中研究最多且最有用的凝集素之一。由于 WGA 与糖缀合物结合，其衍生物和缀合物广泛用于标记细胞膜和纤维化疤痕组织，以进行荧光成像和分析。 WGA 的碳水化合物结合特异性针对 β-1,4-GlcNAc 连接残基（壳糊精）的序列。每个单体包含两个相同的、非相互作用的结合位点，与 3 或 4 个 β-1,4-GlcNAc 单元互补。在所检查的单糖中，只有 GlcNAc 与 WGA 结合。 ManNAc 不结合，GalNAc 仅微弱结合。 WGA 的 iFluor®647 缀合物表现出 iFluor® 647 染料的明亮红色荧光。 iFluor®647 WGA 缀合物与 AF647 WGA 缀合物一样结合唾液酸和 N-乙酰氨基葡萄糖残基。

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样品分析方案

溶液配制

储备溶液配制 

iFluor 647 小麦胚芽凝集素 (WGA) 缀合物储备液 (200X)

将 500 µL ddH2O 添加到粉末小瓶中，制成 2 mg/mL 的储备溶液。 注意：重新配制的缀合物溶液可在 2-8 °C 下短期储存，或在 -20 °C 下长期储存。

工作溶液配制 

iFluor 647-小麦胚芽凝集素 (WGA) 缀合物​​工作溶液 (1X)

将 5 μL 200X WGA 储备溶液添加到 1 mL HHBS 缓冲液中。
注意：不同细胞系的优化染色浓度可能不同。 活细胞的推荐起始浓度为 5-10 µg/mL。

操作步骤

1.活细胞染色
1.1 用 HHBS 缓冲液清洗细胞两次。
1.2 添加 100 µL iFluor 647-WGA 工作溶液。
1.3 将细胞与 WGA 工作溶液在 37°C 下孵育 10-30 分钟。
1.4 用 HHBS 缓冲液清洗细胞两次。
1.5 使用 Cy5 滤光片组在荧光显微镜上成像细胞。

2.固定细胞染色
2.1 在PBS中使用4％的甲醛固定细胞。
注意：对于固定细胞膜染色，建议在没有透化步骤的情况下进行染色。固定后透化步骤会导致细胞内区室染色，如高尔基体和内质网 (ER) 结构染色。
2.2 添加 100 µL iFluor 647-WGA 工作溶液。
2.3 在室温下用 WGA 工作溶液孵育细胞 10-30 分钟。
2.4 用 HHBS 缓冲液清洗细胞两次。
2.5 使用 Cy5 滤光片组在荧光显微镜上成像细胞。

图示

图 1. 使用 iFluor 647-小麦胚芽凝集素 (WGA) 缀合物​​以 5 µg/mL的浓度在活细胞（Hela细胞） 染色 30 分钟，然后用 Hoechst 33342（Cat# 17535）染色。使用带Cy5和DAPI滤波片组的荧光显微镜成像。

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