使用差异定位SunTag组件实现对细胞质内高含量蛋白的活细胞观察

生物过程由数百万个蛋白质驱动.蛋白质结构和蛋白质定位是蛋白质行使功能的关键.在过去的研究中,快速发展的荧光显微镜成像技术使研究人员可以直接观察蛋白质在活细胞内的定位.然而,对细胞质内高含量蛋白的活细胞观察仍然存在技术障碍.我们通过改进SunTag标记技术,使用差异定位的SunTag组件:一方面,在研究目的蛋白(POI)上标记多个V4抗原;另一方面,将识别V4抗原的抗体GCN4融合的绿色荧光蛋白(GCN4-GFP)通过差异定位后限制其与抗原的结合量.我们的方法大大降低了背景荧光信号,实现了对dynamin膜上功能单位的直接可视化.

Visualize Cytoplasmic Abundant Protein Localization via Sun Tag Strategy

Biological processes are powered by millions of proteins.Both protein structure and protein localization are critical for protein function.In the past half-decade,development in fluorescence microscopy enables scientists to directly study protein localization.However,it remains challenging to directly visualize abundant protein localization lively.Here,we present a method to visualize cytoplasmic abundant protein localization in living cells.We labeled the protein of interest (POI) by fusion with SunTag,which can recruit multiple copies of antibody fused green fluorescent protein (GCN4-GFP).We limited the amount of GCN4-GFP by mis-localization,greatly reduced background signal and achieved directly visualization of POI at a resolution of its functional unit.As a proof of principle,we characterize the localization of dynamin.