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Open Access Green as soon as Postprint is submitted to ZB.
Single- and dual-parameter FRET kinase probes based on pleckstrin.
Nat. Protoc. 1, 1044-1055 (2006)
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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Publication type
Article: Journal article
Document type
Scientific Article
ISSN (print) / ISBN
1754-2189
e-ISSN
1750-2799
Journal
Nature Protocols
Quellenangaben
Volume: 1,
Issue: 2,
Pages: 1044-1055
Publisher
Nature Publishing Group
Reviewing status
Peer reviewed
Institute(s)
Institute of Structural Biology (STB)