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Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry

Nature Protocols volume 3, pages 2233 (2008) | Download Citation

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Abstract

We present a high-throughput approach to study weak protein–protein interactions by coupling bimolecular fluorescent complementation (BiFC) to flow cytometry (FC). In BiFC, the interaction partners (bait and prey) are fused to two rationally designed fragments of a fluorescent protein, which recovers its function upon the binding of the interacting proteins. For weak protein–protein interactions, the detected fluorescence is proportional to the interaction strength, thereby allowing in vivo discrimination between closely related binders with different affinity for the bait protein. FC provides a method for high-speed multiparametric data acquisition and analysis; the assay is simple, thousands of cells can be analyzed in seconds and, if required, selected using fluorescence-activated cell sorting (FACS). The combination of both methods (BiFC-FC) provides a technically straightforward, fast and highly sensitive method to validate weak protein interactions and to screen and identify optimal ligands in biologically synthesized libraries. Once plasmids encoding the protein fusions have been obtained, the evaluation of a specific interaction, the generation of a library and selection of active partners using BiFC-FC can be accomplished in 5 weeks.

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Acknowledgements

We thank Manuela Costa and Jaume Comas for technical assistance in the FC technique. This work has been supported by grants GEN2003-20642 and BIO2004-05879 (MEC, Spain), by LSHG.2006-018830-CAMP (EC-Dir. F), and by CeRBA and SGR00037 (Generalitat de Catalunya).

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Affiliations

  1. Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.

    • Montse Morell
    • , Francesc Xavier Aviles
    •  & Salvador Ventura
  2. Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, E-08193 Bellaterra, Spain.

    • Alba Espargaro
    • , Francesc Xavier Aviles
    •  & Salvador Ventura

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Correspondence to Salvador Ventura.

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https://doi.org/10.1038/nprot.2007.496

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