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

[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

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