Abstract：
:A complete understanding of signaling processes at the plasma membrane depends on a quantitative characterization of the interactions of the involved proteins. Fluorescence recovery after photobleaching (FRAP) is a widely used and convenient technique to obtain kinetic parameters on protein interactions in living cells. FRAP experiments to determine unbinding time constants for proteins at the plasma membrane, however, are often hampered by non-specific contributions to the fluorescence recovery signal. On the example of the interaction between the T cell receptor (TCR) and the Syk kinase ZAP70, we present here an approach based on protein micropatterning that allows the elimination of such non-specific contributions and considerably simplifies analysis of FRAP data. Specifically, detection and reference areas are created within single cells, each being either enriched or depleted in TCR, which permits the isolation of ZAP70-TCR binding in a straight-forward manner. We demonstrate the applicability of our method by comparing it to a conventional FRAP approach.
journal_name
Sci Repjournal_title
Scientific reportsauthors
Motsch V,Brameshuber M,Baumgart F,Schütz GJ,Sevcsik Edoi
10.1038/s41598-019-39865-0subject
Has Abstractpub_date
2019-03-01 00:00:00pages
3288issue
1issn
2045-2322pii
10.1038/s41598-019-39865-0journal_volume
9pub_type
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