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

doi:10.11983/CBB19143

Abstract

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

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

Add to citation manager EndNote|Ris|BibTeX

URL: https://www.chinbullbotany.com/EN/10.11983/CBB19143

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

Figures/Tables 4

References 7

[1]	Fernández-Bautista N, Fernández-Calvino L, Muñoz A, Castellano MM (2017). HOP3, a member of the HOP family in Arabidopsis, interacts with BiP and plays a major role in the ER stress response. Plant Cell Environ 40, 1341-1355.
[2]	Hirsch S, Kim J, Muñoz A, Heckmann AB, Downie JA, Oldroyd GED (2009). GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula. Plant Cell 21, 545-557.
[3]	Muñoz A, Mangano S, González-García MP, Contreras R, Sauer M, De Rybel B, Weijers D, Sánchez-Serrano JJ, Sanmartin M, Rojo E (2017). RIMA-dependent nuclear accumulation of IYO triggers auxin-irreversible cell differentiation in Arabidopsis. Plant Cell 29, 575-588.
[4]	Smith DB, Johnson KS (1988). Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31-40.
[5]	Stasi M, De Luca M, Bucci C (2015). Two-hybrid-based systems: powerful tools for investigation of membrane traffic machineries. J Biotechnol 202, 105-117.
[6]	Takahashi Y (2015). Co-immunoprecipitation from transfected cells. In: Meyerkord CL, Fu HA, eds. Protein-Protein Interactions. York: Humana Press.pp. 381-389.
[7]	Wissmueller S, Font J, Liew CW, Cram E, Schroeder T, Turner J, Crossley M, Mackay JP, Matthews JM (2011). Protein-protein interactions: analysis of a false positive GST pulldown result. Proteins 79, 2365-2371.

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)

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)

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

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

[1]	Yun Han, Xiaofeng Chi, Jingya Yu, Xujie Ding, Shilong Chen, Faqi Zhang. A checklist of wild vascular plants in Qinghai, China [J]. Biodiv Sci, 2023, 31(9): 23280-.
[2]	Yousheng Chen, Zhuqiu Song, Ran Wei, Yan Luo, Wenli Chen, Fusheng Yang, Lianming Gao, Yuan Xu, Zhuoxin Zhang, Pengcheng Fu, Chunlei Xiang, Huanchong Wang, Jiachen Hao, Shiyong Meng, Lei Wu, Bo Li, Shengxiang Yu, Shuren Zhang, Li He, Xinqiang Guo, Wenguang Wang, Yihua Tong, Qi Gao, Wenqun Fei, Youpai Zeng, Lin Bai, Zichao Jin, Xingjie Zhong, Buyun Zhang, Siyi Du. A dataset on inventory and geographical distribution of vascular plants in Xizang, China [J]. Biodiv Sci, 2023, 31(9): 23188-.
[3]	Zhuqiu Song, Wen Ye, Shiyong Dong, Zichao Jin, Xingjie Zhong, Zhen Wang, Buyun Zhang, Yechun Xu, Wenli Chen, Shijin Li, Gang Yao, Zhoufeng Xu, Shuai Liao, Yihua Tong, Youpai Zeng, Yunbao Zeng, Yousheng Chen. A dataset on inventory and geographical distributions of higher plants in Guangdong, China [J]. Biodiv Sci, 2023, 31(9): 23177-.
[4]	Shifeng Wen, Zhihua Zhou, Tuo He, Hui Dong, Liangchen Yuan, Zeyang Lu, Yongteng Wang, Lin Guo, Jiangping Shu, Kaifan Li. Formulating the National Botanical Garden System Layout Plan of China: Background, procedures, ideas and key considerations [J]. Biodiv Sci, 2023, 31(9): 23193-.
[5]	Yong Li, Sanqing Li, Huan Wang. A dataset of wild vascular plants and their distributions in Tianjin, China [J]. Biodiv Sci, 2023, 31(9): 23128-.
[6]	Tingting Deng, Yan Wei, Siyuan Ren, Yan Zhu. Effects of topography and stand structure of warm temperate deciduous broad-leaved forest on understory herb diversity in Donglingshan, Beijing [J]. Biodiv Sci, 2023, 31(7): 22671-.
[7]	Xinjing Wu, Jinfeng Chen, Guofa Cui. Proposals for updating the List of National Key Protected Wild Plants—Based on an analysis of existing conservation lists [J]. Biodiv Sci, 2023, 31(7): 22622-.
[8]	Caiqun Liang, Yukai Chen, Xiaobo Yang, Kai Zhang, Donghai Li, Yuexin Jiang, Jinghan Li, Chongyang Wang, Shunwei Zhang, Zicheng Zhu. A dataset on inventory and geographical distributions of wild vascular plants in Hainan Province, China [J]. Biodiv Sci, 2023, 31(6): 23067-.
[9]	Chang An, Yixue Zhuang, Ping Zheng, Yanxiang Lin, Chengzi Yang, Yuan Qin. A checklist of vascular plants in Fujian Province, China [J]. Biodiv Sci, 2023, 31(6): 22537-.
[10]	Cheng Du, Yuan Wang, Xiaoling Yan, Jing Yan, Huiru Li, Qingfei Zhang, Yonghong Hu. Composition and historical changes of plant species diversity in Shanghai and the updated checklist of Shanghai vascular plants (2022) [J]. Biodiv Sci, 2023, 31(6): 23093-.
[11]	Yi Li, Xi Zhang, Yanhui Yuan, Pichang Gong, Jinxing Lin. Aptamers and Their Applications in Plant Science Researches [J]. Chinese Bulletin of Botany, 2023, 58(6): 935-945.
[12]	Zhaoyang Jing, Keguang Cheng, Heng Shu, Yongpeng Ma, Pingli Liu. Whole genome resequencing approach for conservation biology of endangered plants [J]. Biodiv Sci, 2023, 31(5): 22679-.
[13]	Yu Xiao, Yuran Li, Hexiang Duan, Zhengtao Ren, Shengbi Feng, Zhicheng Jiang, Jiahua Li, Pin Zhang, Jinming Hu, Yupeng Geng. Invasion status and control measures for alien plants within the Gaoligong Mountains [J]. Biodiv Sci, 2023, 31(5): 23011-.
[14]	Yaochu Sun, Yuanfei Pan, Mu Liu, Xiaoyun Pan. The specialist-to-generalist ratio affects growth and defense strategy of invasive plant Alternanthera philoxeroides [J]. Biodiv Sci, 2023, 31(4): 22632-.
[15]	Yexi Zhao, Jiayu Zhang, Zihan Li, Qinmijia Xie, Xin Deng, Nan Wang. Use of native and alien plants during night roosting by urban birds in Beijing [J]. Biodiv Sci, 2023, 31(3): 22399-.