UNIT 14.10 In Vivo Imaging of Signal Transduction Cascades with Probes Based on Förster Resonance Energy Transfer (FRET)
Published Online: 1 DEC 2009
Copyright © 2009 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Cell Biology
How to Cite
Nakamura, T. and Matsuda, M. 2009. In Vivo Imaging of Signal Transduction Cascades with Probes Based on Förster Resonance Energy Transfer (FRET). Current Protocols in Cell Biology. 45:14.10:14.10.1–14.10.12.
- Published Online: 1 DEC 2009
- Published Print: DEC 2009
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. Curr. Protoc. Cell Biol. 45:14.10.1-14.10.12. © 2009 by John Wiley & Sons, Inc.
- unimolecular probe;
- Ras GTPase;
- Rho GTPase