Espargaro, Alba

Morell, Montse

Repositorio Institucional de la Universidad de Córdoba

Posters S4. Protein Interactions and Protein Arrays109P. 43MONITORING iN ViVO PROTEIN-PROTEIN INTERACTIONS BY COUPLING BIMOLECULAR FLUORESCENCE COMPLEMENTATION (BIFC) AND FLOW CYTOMETRYMontse Morell, Alba Espargaro, Francesc X. Avilés and Salvador VenturaInstitut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, E-08193 Cerdanyola del Valles (Spain)We present a high-throughput approach to study weak protein-protein interactions by coupling bimolecular fluorescent complementation (BiFC) to flow cytometry (FC). Bimolecular Fluorescence Complementation (BIFC) is a method to detect protein-protein interactions based on the formation of a fluorescent complex by fragments of the yellow fluorescent protein (NYFP and CYFP) brought together by the association of two interaction partners fused to the fragments1. 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 protein2,3. 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.On the other hand, the combination of both technologies has also been applied to detect the interference of protein interactions4. In a test case, we demonstrate that the inhibition of protein interactions results in a concomitant decrease in fluorescence emission. It has to be taken into account that the identification of molecules able to modulate protein contacts is of significant interest for drug discovery and chemical biology. Therefore, the combination of BIFC with flow cytometry might provide an effective means to detect interaction modulators among large chemical or peptidic libraries by directly reading out changes in the reporter signal.1. Hu CD, Grinberg AV, Kerppola TK. (2006) Curr Protoc Cell Biol. Jan 21:Unit 21.32. Morell M, Espargaro A, Aviles, FX, Ventura, S (2007) Proteomics 7, 1023-36.3. Morell M., Espargaro A., Aviles FX., Ventura S (2008) Nature Protocols 3, 22-334. Morell M, Czihal P, Hoffmann R, Otvos L, Avilés FX, Ventura S (2008) Proteomics 8: 3433-42.

Monitoring in vivo protein-protein interactions by coupling bimolecular fluorescence complemetation (BIFC) and flow cytometry

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Repositorio Institucional de la Universidad de Córdoba