Liang Jun, De Castro Aijo, Flores Lizette
Department of Science, Borough of Manhattan Community College/CUNY;
Department of Science, Borough of Manhattan Community College/CUNY.
J Vis Exp. 2018 Jul 30(137):57914. doi: 10.3791/57914.
In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and γ-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.
在本实验方案中，绿色荧光蛋白（GFP）融合蛋白和4',6-二脒基-2-苯基吲哚（DAPI）染色用于追踪蛋白质亚细胞定位变化；特别是在热应激条件下的核转位。蛋白质会对外部和内部信号做出相应反应。一种常见机制是改变其亚细胞定位。本文介绍了一种无需抗体、放射性标记或共聚焦显微镜即可追踪蛋白质定位的实验方案。在本文中，GFP用于标记秀丽隐杆线虫中的目标蛋白EXL-1，它是氯化物细胞内通道蛋白（CLICs）家族的成员之一，包括哺乳动物的CLIC4。通过转化和γ辐射创建了一个整合的翻译exl-1::gfp转基因品系（带有启动子和完整基因序列），并稳定表达该基因和gfp。最近的研究表明，在热应激而非氧化应激下，EXL-1::GFP会在细胞核中积累。将GFP信号与细胞核结构和DAPI信号重叠，可确认应激条件下EXL-1的亚细胞定位变化。本实验方案介绍了两种用于DAPI染色的不同固定方法：乙醇固定和丙酮固定。本文介绍的DAPI染色实验方案快速高效，既能保留GFP信号，又能保留蛋白质亚细胞定位变化。该方法仅需一台配备Nomarski、FITC滤光片和DAPI滤光片的荧光显微镜。它适用于小型实验室环境、本科生研究、高中生研究以及生物技术课堂。
