Luciferase Complementation Assay for Protein-Protein Interactions in Plants

Q1 Agricultural and Biological Sciences

Current protocols in plant biology Pub Date : 2018-04-02 DOI:10.1002/cppb.20066

Zhaoyang Zhou, Guozhi Bi, Jian-Min Zhou

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引用次数: 52

Abstract

Constitutive and dynamic protein-protein interactions are fundamental to all aspects of cellular processes. Compared to other techniques measuring protein-protein interactions in plants, the luciferase complementation assay has a number of advantages: it detects plant protein-protein interactions in real time, requires little hands-on manipulation of samples, is highly quantitative, has extremely low background, and can be easily scaled up for high-throughput interactome studies. Here, we describe a protocol that includes two alternate data collection methods to quantify luminescence results based on Agrobacterium-mediated transient luciferase expression in Nicotiana benthamiana. One data collection method employs a charge-coupled device imaging system that allows the interactions to be presented as images, and the other employs a luminometer, which enables the assay to be conducted in a 96-well plate. Technical parameters related to frequently encountered problems and common errors, presented here, are important for performing this assay successfully. © 2018 by John Wiley & Sons, Inc.

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植物蛋白-蛋白相互作用的荧光素酶互补试验

构成的和动态的蛋白质-蛋白质相互作用是细胞过程的所有方面的基础。与测量植物中蛋白质-蛋白质相互作用的其他技术相比，荧光素酶互补分析具有许多优点:它可以实时检测植物蛋白质-蛋白质相互作用，几乎不需要对样品进行动手操作，高度定量，具有极低的背景，并且可以很容易地扩大用于高通量相互作用组研究。在这里，我们描述了一种方案，其中包括两种替代的数据收集方法，以基于农杆菌介导的瞬时荧光素酶表达来量化benthamiana中的发光结果。一种数据收集方法采用电荷耦合设备成像系统，该系统允许相互作用以图像的形式呈现，另一种方法采用光度计，该方法使分析能够在96孔板中进行。这里介绍的与经常遇到的问题和常见错误相关的技术参数对于成功执行该分析很重要。©2018 by John Wiley &儿子,Inc。

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来源期刊

Current protocols in plant biology Agricultural and Biological Sciences-Plant Science

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期刊介绍： Sound and reproducible laboratory methods are the foundation of scientific discovery. Yet nuances that are critical for an experiment''s success are not captured in the primary literature but exist only as part of a lab''s oral tradition. Current Protocols in Plant Biology provides reproducible step-by-step instructions for protocols relevant to plant research. Furthermore, Current Protocols content is thoughtfully organized by topic for optimal usage and to maximize contextual knowledge. Quarterly issues allow Current Protocols in Plant Biology to constantly evolve to keep pace with the newest discoveries and developments. Current Protocols in Plant Biology is the comprehensive source for protocols in the multidisciplinary field of plant biology, providing an extensive range of protocols from basic to cutting edge. Coverage includes: Extraction and analysis of DNA, RNA, proteins Chromosome analysis Transcriptional analysis Protein expression Metabolites Plant enzymology Epigenetics Plant genetic transformation Mutagenesis Arabidopsis, Maize, Poplar, Rice, and Soybean, and more.