Design and Optimization of Genetically Encoded Fluorescent Biosensors: GTPase Biosensors

Louis Hodgson, Olivier Pertz, Klaus M. Hahn

Research output: Chapter in Book/Report/Conference proceedingChapter

44 Scopus citations

Abstract

This chapter details the design and optimization of biosensors based on a design used successfully to study nucleotide loading of small GTPase proteins in living cells. This design can be generalized to study many other protein activities, using a single, genetically encoded chain incorporating the protein to be studied, an "affinity reagent" which binds only to the activated form of the targeted protein, and mutants of the green fluorescent protein (GFP) that undergo fluorescence resonance energy transfer (FRET). Specific topics include procedures and caveats in the design and cloning of single-chain FRET sensors, in vitro and in vivo validation, expression in living cell systems for biological studies, and some general considerations in quantitative fluorescence imaging.

Original languageEnglish (US)
Title of host publicationFluorescent Proteins
EditorsKevin Sullivan
Pages63-81
Number of pages19
DOIs
StatePublished - Jan 1 2008
Externally publishedYes

Publication series

NameMethods in Cell Biology
Volume85
ISSN (Print)0091-679X

ASJC Scopus subject areas

  • Cell Biology

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    Hodgson, L., Pertz, O., & Hahn, K. M. (2008). Design and Optimization of Genetically Encoded Fluorescent Biosensors: GTPase Biosensors. In K. Sullivan (Ed.), Fluorescent Proteins (pp. 63-81). (Methods in Cell Biology; Vol. 85). https://doi.org/10.1016/S0091-679X(08)85004-2