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Rapid development of genetically encoded FRET reporters.

Research article Created on 15 Jan 2013

Authors

Alen Piljić, Iñaki de Diego, Matthias Wilmanns, Carsten Schultz

Published in

ACS chemical biology. Volume 6. Issue 7. Pages 685-91. Jul 14, 2011. Epub Apr 19, 2011.

Abstract

To meet the demand on genetically encoded reporter molecules for live cell imaging, we introduce a new facile combined cloning and FRET reporter analysis strategy. The versatile and fully orthogonal cloning approach involves a set of up to 36 vectors featuring a variety of fluorescent protein FRET pairs and different length linkers. The construct set was successfully applied to two calmodulin-binding proteins, the death-associated protein kinase 1 (DAPK1) and calcium/calmodulin-dependent protein kinase II α (Camk2a). Clone analysis and reporter validation was performed by printing plasmid DNA arrays and subsequent semiautomated microscopy of reversely transfected cells. Characterization of the best performing DAPK1 and Camk2a reporters revealed significant differences in translating calcium signals into kinase responses despite the close functional and structural similarity.

PMID:
21506563
Bibliographic data and abstract were imported from PubMed on 15 Jan 2013.

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  • Wednesday, 25 January 2017 - 06:05 UTC

    This comment has been removed due to violation of our Terms.

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    Alen Piljić Saturday, 21 February 2015 - 17:39 UTC

    As I understand, the transfection protocol described here has by now been vastly improved. I don't have access to it, but anyone interested should contact the head of the lab. Hopefully they'll be willing to share it.

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    Alen Piljić Friday, 17 May 2013 - 10:55 UTC

    A detailed cloning protocol for cloning of FRET reporters described in this publication can be found here http://www.lifescience.net/protocols/42/cloning-of-ratiometric-fret-sensors-using-f-series/

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