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Int. J. Mol. Sci. 2014, 15(6), 9628-9643; doi:10.3390/ijms15069628
Technical Note

A Cautionary Note on the Use of Split-YFP/BiFC in Plant Protein-Protein Interaction Studies

1
, 1
, 1
 and 1,2,*
1 Plant Research International, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands 2 Physiology of Flower Bulbs, Department of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands
* Author to whom correspondence should be addressed.
Received: 3 April 2014 / Revised: 17 April 2014 / Accepted: 20 May 2014 / Published: 30 May 2014
(This article belongs to the collection Proteins and Protein-Ligand Interactions)
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Abstract

Since its introduction in plants 10 years ago, the bimolecular fluorescence complementation (BiFC) method, or split-YFP (yellow fluorescent protein), has gained popularity within the plant biology field as a method to study protein-protein interactions. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. The major drawback of BiFC is that the fluorescent protein halves are prone to self-assembly independent of a protein-protein interaction event. To circumvent this problem, several modifications of the technique have been suggested, but these modifications have not lead to improvements in plant BiFC protocols. Therefore, it remains crucial to include appropriate internal controls. Our literature survey of recent BiFC studies in plants shows that most studies use inappropriate controls, and a qualitative rather than quantitative read-out of fluorescence. Therefore, we provide a cautionary note and beginner’s guideline for the setup of BiFC experiments, discussing each step of the protocol, including vector choice, plant expression systems, negative controls, and signal detection. In addition, we present our experience with BiFC with respect to self-assembly, peptide linkers, and incubation temperature. With this note, we aim to provide a guideline that will improve the quality of plant BiFC experiments.
Keywords: protein-protein interaction; split-YFP (yellow fluorescent protein); bimolecular fluorescence complementation (BiFC); protein complementation assay (PCA); in planta; fluorescence microscopy protein-protein interaction; split-YFP (yellow fluorescent protein); bimolecular fluorescence complementation (BiFC); protein complementation assay (PCA); in planta; fluorescence microscopy
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Horstman, A.; Tonaco, I.A.N.; Boutilier, K.; Immink, R.G.H. A Cautionary Note on the Use of Split-YFP/BiFC in Plant Protein-Protein Interaction Studies. Int. J. Mol. Sci. 2014, 15, 9628-9643.

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