Design and Optimization of Genetically Encoded Fluorescent Biosensors: GTPase Biosensors

Louis Hodgson, Olivier Pertz, Klaus M. Hahn

Research output: Contribution to journalArticle

42 Citations (Scopus)

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)
Pages (from-to)63-81
Number of pages19
JournalMethods in Cell Biology
Volume85
DOIs
StatePublished - 2008
Externally publishedYes

Fingerprint

GTP Phosphohydrolases
Biosensing Techniques
Fluorescence Resonance Energy Transfer
Proteins
Monomeric GTP-Binding Proteins
Optical Imaging
Mutant Proteins
Green Fluorescent Proteins
Organism Cloning
Nucleotides

ASJC Scopus subject areas

  • Cell Biology

Cite this

Design and Optimization of Genetically Encoded Fluorescent Biosensors : GTPase Biosensors. / Hodgson, Louis; Pertz, Olivier; Hahn, Klaus M.

In: Methods in Cell Biology, Vol. 85, 2008, p. 63-81.

Research output: Contribution to journalArticle

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