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Proximity Ligation Assay for High Content Profiling of Cell Signaling Pathways on a Microfluidic Chip

Blazek, M. and Betz, C. and Reth, M. and Hall, M. N. and Zengerle, R. and Meier, M.. (2013) Proximity Ligation Assay for High Content Profiling of Cell Signaling Pathways on a Microfluidic Chip. Molecular & cellular proteomics, Vol. 12. pp. 3898-3907.

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Official URL: http://edoc.unibas.ch/dok/A6183939

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Abstract

Here, we present the full integration of a proximity ligation assay (PLA) on a microfluidic chip for systematic cell signaling studies. PLA is an in situ technology for detection of protein interaction, post-translational modification, concentration, and cellular location with single molecule resolution. Analytical performance advances on chip are achieved, including full automation of the biochemical PLA steps, target multiplexing, and reduction of antibody consumption by two orders of magnitudes compared to standard procedures. In combination with a microfluidic cell-culturing platform, control over 128 cell culture microenvironments is gained. We demonstrate the use of the combined cell-culture and protein analytic assay on chip by characterizing the Akt signaling pathway upon PDGF stimulation. Signal transduction is detected by monitoring the phosphorylation state of Akt, GSK-3β, p70S6K, S6, Erk1/2, mTOR, and the cellular location of FoxO3a in parallel with the PLA. Single-cell PLA results revealed for Akt and direct targets of Akt a maximum activation time of 4-8 min upon PDGF stimulation. Activation times for phosphorylation events downwards in the Akt signaling pathway including phosphorylation of S6, p70S6K, and mTOR are delayed by 8-10 min or exhibit a response time of at least 1 h. Quantitative confirmation of the Akt phosphorylation signal was determined with the help of a mouse embryonic fibroblast cell line (MEF) deficient for rictor. Taken together, the miniaturized PLA chip establishes a biotechnological tool for general cell signaling studies and their dynamics relevant for a broad range of biological inquiry.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Hall)
UniBasel Contributors:Hall, Michael N.
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:The American Society for Biochemistry and Molecular Biology
ISSN:1535-9484
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:20 Jun 2014 07:56
Deposited On:20 Jun 2014 07:56

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