蛋白互作在细胞生命活动中发挥关键作用, 在不同时空层面上参与多种细胞学过程, 因此研究蛋白互作对理解分子调控网络至关重要。通常情况下, 利用酵母双杂交系统筛选植物蛋白互作必须通过体外和体内系统进行验证。Pull-down和Co-IP是验证植物蛋白互作的常用技术。Pull-down被广泛用于体外验证蛋白间的直接互作; 而在植物活体内, 利用本氏烟草(Nicotiana benthamiana)叶片瞬时表达蛋白, 继而通过Co-IP进行鉴定是目前验证蛋白互作最简单且最有效的方法之一。该文对GST Pull-down和烟草瞬时表达系统中Co-IP技术原理及实验方案进行详细描述, 以期为验证植物蛋白互作提供参考。

Protein-protein interactions play a key role in cellular signaling, involved in various biological processes. Studies on these interactions are therefore crucial toward understanding the regulatory networks of cellular signaling. It is a standard practice that the protein-protein interactions identified by the yeast two-hybrid system should be independently confirmed by in vitro and in vivo approaches. Pull-down and co-immunoprecipitation (Co-IP) are routine approaches to detect protein-protein interactions. Pull-down assay is used to detect direct or physical interactions between proteins in vitro. In plant biology studies, one of the most convenient methods to detect protein-protein interactions is the transient expression of the target proteins in Nicotiana benthamiana leaves followed by the Co-IP assay. In this paper, we describe the principles and protocols for the GST tag-based pull-down assay and the Co-IP assay of proteins transiently expressed in N. benthamiana leaves, providing a reference for detecting plant protein-protein interactions.