植物中验证蛋白相互作用的Pull-down和Co-IP技术

doi:10.11983/CBB19143

植物学报 ›› 2020, Vol. 55 ›› Issue (1): 62-68.DOI: 10.11983/CBB19143

植物中验证蛋白相互作用的Pull-down和Co-IP技术

徐重益()

中国科学院植物研究所, 中国科学院分子植物科学卓越创新中心, 植物分子生理学重点实验室, 北京 100093

收稿日期:2019-08-01 接受日期:2019-08-30 出版日期:2020-01-01 发布日期:2019-12-20
通讯作者: 徐重益
基金资助:
国家自然科学基金面上项目(No. 31371447);国家自然科学基金面上项目(No. 31771632)

Pull-down and Co-immunoprecipitation Assays of Interacting Proteins in Plants

Chongyi Xu()

Key Laboratory of Plant Molecular Physiology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

Received:2019-08-01 Accepted:2019-08-30 Online:2020-01-01 Published:2019-12-20
Contact: Chongyi Xu

摘要/Abstract

摘要： 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。

关键词: 植物, 蛋白互作, Pull-down, Co-IP

Abstract: 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.

Key words: plants, protein-protein interactions, Pull-down, Co-immunoprecipitation

徐重益. 植物中验证蛋白相互作用的Pull-down和Co-IP技术. 植物学报, 2020, 55(1): 62-68.

Chongyi Xu. Pull-down and Co-immunoprecipitation Assays of Interacting Proteins in Plants. Chinese Bulletin of Botany, 2020, 55(1): 62-68.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB19143

https://www.chinbullbotany.com/CN/Y2020/V55/I1/62

图/表 4

表1 GST Pull-down实验流程中所使用的相关试剂配方

Table 1 Related reagent formulations used in the GST Pull- down

Solution	Composition	Final concentration
1× PBS buffer (pH7.4)	NaCl KCl	137 mmol·L^-1 2.7 mmol·L^-1
	Na₂HPO₄	10 mmol·L^-1
	KH₂PO₄	2 mmol·L^-1
NETN buffer	Tris-HCl (pH8.0)	20 mmol·L^-1
	NaCl	100 mmol·L^-1
	EDTA	0.5 mmol·L^-1
	Nonidet P-40 (NP-40)	0.5% (v/v)
3× SDS sam- ple buffer	Tris-HCl (pH6.8) Glycerin	167 mmol·L^-1 33% (v/v)
	Sodium dodecyl sulfate	6.6% (w/v)
	Bromophenol blue	0.01% (w/v)
	β-mercaptoethanol	7.5% (v/v)

表2 Co-IP实验流程中所使用的相关试剂配方

Table 2 Related reagent formulations used in the Co-IP

Solution	Composition	Final concentration
Infiltration buffer	MES-KOH (pH5.6)	10 mmol·L^-1
	MgCl₂	10 mmol·L^-1
	Acetyl syringone (AS)	0.2 mmol·L^-1
NB1 buffer	Tris-MES (pH8.0)	50 mmol·L^-1
	Sucrose	500 mmol·L^-1
	MgCl₂	1 mmol·L^-1
	EDTA	10 mmol·L^-1
	DTT	5 mmol·L^-1
	PMSF	1 mmol·L^-1
	Cocktail	100×
3× SDS sample buffer	Same as Pull-down

图1 利用原核表达系统检测融合GST ( 谷胱甘肽-S-转移酶)的诱饵蛋白与捕获蛋白互作的Pull-down实验流程图 IPTG: 异丙基硫代-β-D-半乳糖苷; GSH: 谷胱甘肽

Figure 1 Schematic representation of the Pull-down procedure in prokaryotic expression system to test the interaction between a bait protein tagged with GST (glutathione S-transferase) and a prey protein IPTG: Isopropyl-β-D-thiogalactopyranoside; GSH: Glutathione

图2 利用烟草瞬时转化体系检测融合Myc的诱饵蛋白与融合Flag的捕获蛋白互作的Co-IP实验流程图

Figure 2 Schematic representation of the Co-immunoprecipitation procedure from infiltrated Nicotiana benthamiana leaves to test the interaction between a prey protein tagged with Flag and a bait protein tagged with Myc

参考文献 7

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植物中验证蛋白相互作用的Pull-down和Co-IP技术

Pull-down and Co-immunoprecipitation Assays of Interacting Proteins in Plants

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