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Analysis of Signaling Events by Dynamic Phosphoflow Cytometry

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.

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Classifications


Resource Type: Bibliography, Diagram, Illustration, Journal article/Issue, Laboratory manual
Audience Level: Undergraduate upper division 15-16, Graduate, Professional (degree program)

Author and Copyright


Authors and Editors: Guylene Firaguay of Centre de Recherche en Cancerologie de Marseille, France, Jacques A. Nunes of Centre de Recherche en Cancerologie de Marseille, France
Publisher: American Association for the Advancement of Science
Format: application/pdf, image/gif, image/jpeg, text/html
Copyright and other restrictions: Yes
Cost: Yes

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Collection:
STKE/Science Signaling


     
   

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