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Protein Fragment Bimolecular Fluorescence Complementation Analyses for the In vivo Study of Protein-Protein Interactions and Cellular Protein Complex Localizations

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Arabidopsis Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1062))

Abstract

The analyses of protein-protein interactions are crucial for understanding cellular processes including signal transduction, protein trafficking, and movement. Protein fragment complementation assays are based on the reconstitution of protein function when non-active protein fragments are brought together by interacting proteins that were genetically fused to these protein fragments. Bimolecular fluorescence complementation (BiFC) relies on the reconstitution of fluorescent proteins and enables both the analysis of protein-protein interactions and the visualization of protein complex formations in vivo.

Transient expression of proteins is a convenient approach to study protein functions in planta or in other organisms and minimizes the need for time-consuming generation of stably expressing transgenic organisms. Here we describe protocols for BiFC analyses in Nicotiana benthamiana and Arabidopsis thaliana leaves transiently transformed by Agrobacterium infiltration. Further, we discuss different BiFC applications and provide examples for proper BiFC analyses in planta.

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Abbreviations

BiFC:

Bimolecular fluorescence complementation

BiLC:

Bimolecular luminescence complementation

BRET:

Bioluminescence resonance energy transfer

CBB:

Coomassie brilliant blue

CFP:

Cyan FP

coBiFC:

Co-localization of BiFC complexes

ECL:

Enhanced chemiluminescence

FP:

Fluorescent protein

FPC :

FP C-terminal fragment

FPN :

FP N-terminal fragment

FRET:

Fluorescence resonance energy transfer

GFP:

Green FP

iBiSC:

Isolation of BiFC-stabilized complexes

mcBiFC:

Multicolor BiFC

OD600 :

Optical density at 600 nm

PCA:

Protein fragment complementation assay

PPI:

Protein-protein interaction

PVDF:

Polyvinylidene fluoride

RFP:

Red FP

RLuc:

Renilla reniformis luciferase

SUS:

Split-ubiquitin system

TEV:

Tobacco etch virus protease

UbFC:

Ubiquitin-mediated fluorescence complementation

Y2H:

Yeast-two-hybrid

YFP:

Yellow FP

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Acknowledgements

We thank Alex Costa for critical suggestions with the Arabidopsis infiltration protocol, Katrin Held for providing the YC-ABI1 construct, and Jan Niklas Offenborn for providing the OST1 construct. This work was supported by a Feodor Lynen fellowship from the Alexander von Humboldt-Foundation to R.W., a National Institutes of Health grant R01GM060396 and a National Science Foundation grant MCB0918220 to J.I.S., and grants from the DFG (SFB629 and FOR964) to J.K.

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Authors and Affiliations

  1. Division of Biological Sciences, Cell and Developmental Biology Section and Center for Food and Fuel for the 21st Century, University of California San Diego, La Jolla, CA, USA

    Rainer Waadt & Julian I. Schroeder

  2. Molekulargenetik und Zellbiologie der Pflanzen, Institut für Biologie und Biotechnologie der Pflanzen, Universität Münster, Münster, Germany

    Kathrin Schlücking & Jörg Kudla

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  1. Rainer Waadt
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  2. Kathrin Schlücking
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  3. Julian I. Schroeder
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  4. Jörg Kudla
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Editors and Affiliations

  1. CSIC Centro Nacional de Biotecnología, Madrid, Spain

    Jose J. Sanchez-Serrano

  2. Depto. Biologia de Plantas, CSIC Madrid Centro de Investigaciones Biologicas, Madrid, Spain

    Julio Salinas

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Waadt, R., Schlücking, K., Schroeder, J.I., Kudla, J. (2014). Protein Fragment Bimolecular Fluorescence Complementation Analyses for the In vivo Study of Protein-Protein Interactions and Cellular Protein Complex Localizations. In: Sanchez-Serrano, J., Salinas, J. (eds) Arabidopsis Protocols. Methods in Molecular Biology, vol 1062. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-580-4_33

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  • DOI: https://doi.org/10.1007/978-1-62703-580-4_33

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-579-8

  • Online ISBN: 978-1-62703-580-4

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