Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
5897776 | Cytokine | 2013 | 14 Pages |
Abstract
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Keywords
Confocal fluorescence spectroscopyIFN-αTFPCFSORFECFPeGFPOFPSTFPEYFPPEIIFN-γJanus kinaseenhanced cyan fluorescent proteinFluorescence resonance energy transferFRETinterferonIFNInterferon-αinterferon-γEquilibriumopen reading frameFluorescent proteinenhanced yellow fluorescent proteinenhanced green fluorescent proteinpolyethyleneimineReceptor
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Authors
Christopher D. Krause, Gina Digioia, Lara S. Izotova, Sidney Pestka,