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  Single- and dual-parameter FRET kinase probes based on pleckstrin

Brumbaugh, J., Schleifenbaum, A., Stier, G., Sattler, M., & Schultz, C. (2006). Single- and dual-parameter FRET kinase probes based on pleckstrin. Nature Protocols, 1(2), 1044-1055. doi:10.1038/nprot.2006.177.

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Item Permalink: http://hdl.handle.net/21.11116/0000-0001-E165-F Version Permalink: http://hdl.handle.net/21.11116/0000-0001-E166-E
Genre: Journal Article

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NatProt_1_2006_1044.pdf (Any fulltext), 275KB
 
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 Creators:
Brumbaugh, J., Author
Schleifenbaum, A., Author
Stier, Gunter1, Author              
Sattler, Michael, Author
Schultz, C., Author
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1Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society, ou_1497700              

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 Abstract: Here we describe protocols for preparing and using fluorescent probes that respond to conformational changes by altered Foerster resonance energy transfer (FRET) efficiencies upon phosphorylation or, in principle, other posttranslational modifications (PTMs). The sensor protein, a truncated version of pleckstrin, is sandwiched between short-wavelength-excitation green fluorescent protein (GFP2) and yellow fluorescent protein (EYFP). As a result of complex conformational changes of the protein upon phosphorylation, the introduction of a second PTM consensus sequence bestows sensitivity to a second modification and yields a dual-parameter probe. The first phase of the protocol lays out the cloning strategy for single- and dual-parameter FRET sensors, including the construction of a versatile platform into which different consensus sequences may be inserted to create diverse probes. Protocols for fluorescence microscopy of the probes in living cells and image processing are also described. Probe preparation takes 7 d; microscopy and image processing take 2 h.

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Language(s): eng - English
 Dates: 2006-08-172006-08-17
 Publication Status: Published in print
 Pages: 12
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 Rev. Type: Peer
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Title: Nature Protocols
  Other : Nat. Protoc.
Source Genre: Journal
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Pages: - Volume / Issue: 1 (2) Sequence Number: - Start / End Page: 1044 - 1055 Identifier: ISSN: 1750-2799
CoNE: https://pure.mpg.de/cone/journals/resource/1000000000223800_1