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Curr Protoc Cell Biol. 2009 Dec;Chapter 14:Unit 14.10. doi: 10.1002/0471143030.cb1410s45.

In vivo imaging of signal transduction cascades with probes based on Förster Resonance Energy Transfer (FRET).

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Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, Japan.


Genetically encoded FRET probes enable us to visualize a variety of signaling events such as protein phosphorylation and G-protein activation in living cells. This unit focuses on FRET probes wherein both the donor and acceptor are fluorescence proteins and incorporated into a single molecule, i.e., a unimolecular probe. Advantages of these probes lie in their easy loading into cells, simple acquisition of FRET images, and clear evaluation of data. We have developed FRET probes for Ras-superfamily GTPases, designated Ras and interacting protein chimeric unit (Raichu) probes. We hereby describe strategies to develop Raichu-type FRET probes, procedures for their characterization, and acquisition and processing of images. Although improvements upon FRET probes are still based on trial-and-error, we provide practical tips for their optimization and briefly discuss the theory and applications of unimolecular FRET probes.

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