蛋白质-蛋白质相互作用的双分子荧光互补（BiFC）分析及活细胞中亚细胞定位的评估

Bimolecular Fluorescence Complementation (BiFC) Analysis of Protein-Protein Interactions and Assessment of Subcellular Localization in Live Cells.

作者信息

Pratt Evan P S, Owens Jake L, Hockerman Gregory H, Hu Chang-Deng

机构信息

Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Drive, West Lafayette, IN, 47907, USA.

出版信息

Methods Mol Biol. 2016;1474:153-70. doi: 10.1007/978-1-4939-6352-2_9.

DOI:10.1007/978-1-4939-6352-2_9

PMID:27515079

Abstract

Bimolecular fluorescence complementation (BiFC) is a fluorescence imaging technique used to visualize protein-protein interactions (PPIs) in live cells and animals. One unique application of BiFC is to reveal subcellular localization of PPIs. The superior signal-to-noise ratio of BiFC in comparison with fluorescence resonance energy transfer or bioluminescence resonance energy transfer enables its wide applications. Here, we describe how confocal microscopy can be used to detect and quantify PPIs and their subcellular localization. We use basic leucine zipper transcription factor proteins as an example to provide a step-by-step BiFC protocol using a Nikon A1 confocal microscope and NIS-Elements imaging software. The protocol given below can be readily adapted for use with other confocal microscopes or imaging software.

摘要

双分子荧光互补（BiFC）是一种用于在活细胞和动物中可视化蛋白质-蛋白质相互作用（PPI）的荧光成像技术。BiFC的一个独特应用是揭示PPI的亚细胞定位。与荧光共振能量转移或生物发光共振能量转移相比，BiFC具有更高的信噪比，这使其得以广泛应用。在这里，我们描述了如何使用共聚焦显微镜检测和定量PPI及其亚细胞定位。我们以碱性亮氨酸拉链转录因子蛋白为例，提供了一个使用尼康A1共聚焦显微镜和NIS-Elements成像软件的BiFC分步方案。下面给出的方案可以很容易地适用于其他共聚焦显微镜或成像软件。