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.

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

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

Figures/Tables 4

References 7

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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

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