English
 
User Manual Privacy Policy Disclaimer Contact us
  Advanced SearchBrowse

Item

ITEM ACTIONSEXPORT

Released

Journal Article

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

MPS-Authors
/persons/resource/persons117899

Stier,  Gunter
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

Fulltext (public)
There are no public fulltexts available
Supplementary Material (public)
There is no public supplementary material available
Citation

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.


Cite as: http://hdl.handle.net/21.11116/0000-0001-E165-F
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.