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Sci. Signal., 1 September 2009
Vol. 2, Issue 86, p. pl3
[DOI: 10.1126/scisignal.286pl3]

PROTOCOLS

Analysis of Signaling Events by Dynamic Phosphoflow Cytometry

Guylène Firaguay1,2,3 and Jacques A. Nunès1,2,3*

1 Institut National de la Santé et de la Recherche Médicale, Unité 891, Centre de Recherche en Cancérologie de Marseille, F-13009 Marseille, France.
2 Institut Paoli-Calmettes, F-13009 Marseille, France.
3 Université de la Méditerranée, F-13007 Marseille, France.

Abstract: Many proteins involved in cell signaling are phosphorylated. To determine the phosphorylation status of these signaling molecules at the single-cell level, we present a protocol for using state-specific antibodies to detect target phosphoproteins with fluorescence measurements by flow cytometry. To improve the signal intensity, a sandwich-labeling method for the analysis of signaling proteins is performed. By comparing the phosphorylation state of proteins in the presence and absence of sodium pervanadate, a nonspecific tyrosine phosphatase inhibitor, we determined the relative amount of tyrosine-phosphorylated protein in the samples, which reflects the activity of the signaling pathway. This dynamic approach, in combination with the signal amplification through a sandwich-labeling method, produces accurate and reproducible measurement of the activity of signaling pathways.

* Corresponding author. E-mail, jacques.nunes{at}inserm.fr

Citation: G. Firaguay, J. A. Nunès, Analysis of Signaling Events by Dynamic Phosphoflow Cytometry. Sci. Signal. 2, pl3 (2009).

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