Shyu Y John, Hiatt Susan M, Duren Holli M, Ellis Ronald E, Kerppola Tom K, Hu Chang-Deng
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-2091, USA.
Nat Protoc. 2008;3(4):588-96. doi: 10.1038/nprot.2008.16.
The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.
双分子荧光互补（BiFC）分析是一种用于可视化和鉴定活细胞中蛋白质相互作用的强大工具。该分析基于蛋白质片段互补的原理，使用源自荧光蛋白的两个无荧光片段。当通过将两个片段分别连接到一对相互作用蛋白中的一个上，使其在活细胞中聚集在一起时，荧光得以恢复。在此，我们提供了一种基于金星（Venus）的BiFC分析方案，用于可视化活线虫秀丽隐杆线虫中的蛋白质相互作用。我们讨论了如何设计合适的秀丽隐杆线虫BiFC克隆载体，以使用诱导型热休克启动子或天然启动子来实现蛋白质相互作用的可视化；通过显微注射将构建体转化到线虫中；以及分析和解释所得数据。当通过热休克诱导BiFC融合蛋白表达时，荧光信号最早可在诱导后30分钟可视化，并在转基因动物中持续24小时。整个过程需要2至3周完成。
