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Blazek, M.* ; Betz, C.* ; Hall, M.N.* ; Reth, M.* ; Zengerle, R.* ; Meier, M.

Proximity ligation assay for high-content profiling of cell signaling pathways on a microfluidic chip.

Mol. Cell. Proteomics 12, 3898-907 (2013)
DOI PMC
Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.
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 the 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 2 orders of magnitude relative to standard procedures. In combination with a microfluidic cell-culturing platform, this technology allows one to gain control over 128 cell culture microenvironments. 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 states of Akt, GSK-3β, p70S6K, S6, Erk1/2, and 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 to 8 min upon PDGF stimulation. Activation times for phosphorylation events downward in the Akt signaling pathway including the phosphorylation of S6, p70S6K, and mTOR are delayed by 8 to 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 deficient for rictor. In sum, this work with a miniaturized PLA chip establishes a biotechnological tool for general cell signaling studies and their dynamics relevant for a broad range of biological inquiry.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Korrespondenzautor
ISSN (print) / ISBN 1535-9476
e-ISSN 1535-9484
Zeitschrift Molecular and Cellular Proteomics
Quellenangaben Band: 12, Heft: 12, Seiten: 3898-907 Artikelnummer: , Supplement: ,
Verlag American Society for Biochemistry and Molecular Biology
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) Helmholtz Pioneer Campus (HPC)