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Ion Channels

Volume 337 of the series Methods in Molecular Biology™ pp 65-77

Spectroscopy-Based Quantitative Fluorescence Resonance Energy Transfer Analysis

  • Jie ZhengAffiliated withDepartment of Physiology and Membrane Biology, University of California at Davis, School of Medicine

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Abstract

The combination of green fluorescent protein mutants and fluorescence resonance energy transfer (FRET) forms a powerful tool for ion channel studies. A key to successful application of green fluorescent protein-based FRET is to reliably separate the FRET signal from various non-FRET fluorescence emissions that coexist in any experimental system. This chapter introduces a FRET quantification method that is based on fluorescence spectroscopic microscopy. Application of this “spectra FRET” method to both the confocal imaging of Xenopus oocytes and the epifluorescence imaging of culture cells is described. The fluorescence intensity ratio measurement, a complementary non-FRET method for identifying the channel subunit stoichiometry, is also discussed.

Key Words

Confocal microscopy epifluorescence microscopy fluorescence intensity ratio fluorescence resonance energy transfer protein-protein interaction spectroscopy stoichiometry