使用荧光标记的单链抗体片段对活酵母细胞中的蛋白质进行增强荧光成像。
Enhanced fluorescent imaging of proteins in live yeast cells using fluorescently labeled scFv.
作者信息
Tsirkas Ioannis, Zur Tomer, Dovrat Daniel, Paleiov Zohar, Ravkaie Lior, Aharoni Amir
机构信息
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel.
Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva 84105, Israel.
出版信息
STAR Protoc. 2023 Jun 2;4(2):102299. doi: 10.1016/j.xpro.2023.102299.
Fluorescent labeling of proteins is a widespread approach for the microscopic examination of protein function, expression, and localization in the cell. Here, we present a protocol for the labeling of hemagglutinin (HA)-tagged protein of interest (POI) with the single-chain antibody (scFv) 2E2 fused to different fluorescent proteins (FPs) in Saccharomyces cerevisiae. We describe steps for expressing 2E2-FP, and HA tagging and labeling of POI. We detail in vivo fluorescent imaging of proteins at different cellular compartments and with diverse expression levels. For complete details on the use and execution of this protocol, please refer to Tsirkas et al. (2022)..
蛋白质的荧光标记是用于在显微镜下检查蛋白质功能、表达及在细胞中定位的一种广泛应用的方法。在此，我们展示了一种在酿酒酵母中用与不同荧光蛋白（FP）融合的单链抗体（scFv）2E2对感兴趣的血凝素（HA）标记蛋白（POI）进行标记的方案。我们描述了表达2E2-FP以及对POI进行HA标记和标记的步骤。我们详细介绍了在不同细胞区室以及不同表达水平下蛋白质的体内荧光成像。有关此方案的使用和执行的完整详细信息，请参考Tsirkas等人（2022年）的文献。
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