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

Justin Brumbaugh, Andreas Schleifenbaum, Gunter Stier, Michael Sattler, Carsten Schultz

Research output: Contribution to journalArticle

9 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1044-1055
Number of pages12
JournalNature Protocols
Volume1
Issue number2
DOIs
Publication statusPublished - Jul 2006
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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