Abstract
In this paper, we provide a general protocol for labeling proteins with the membrane-permeant fluorogenic biarsenical dye fluorescein arsenical hairpin binder–ethanedithiol (FlAsH-EDT2). Generation of the tetracysteine-tagged protein construct by itself is not described, as this is a protein-specific process. This method allows site-selective labeling of proteins in living cells and has been applied to a wide variety of proteins and biological problems. We provide here a generally applicable labeling procedure and discuss the problems that can occur as well as general considerations that must be taken into account when designing and implementing the procedure. The method can even be applied to proteins with expression below 1 pmol mg−1 of protein, such as G protein–coupled receptors, and it can be used to study the intracellular localization of proteins as well as functional interactions in fluorescence resonance energy transfer experiments. The labeling procedure using FlAsH-EDT2 as described takes 2–3 h, depending on the number of samples to be processed.
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Acknowledgements
Research in the authors' laboratories is supported by the US National Institutes of Health, the Howard Hughes Medical Institute, the Deutsche Forschungsgemeinschaft (German Research Foundation) and the European Research Council.
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C.H., G.G., A.Z., S.R.A. and S.T. conducted experiments; C.H. and G.G. drafted the paper; and M.H.E., R.Y.T. and M.J.L. finalized the paper.
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The University of California, San Diego owns a patent on FlAsH, ReAsH, and tetracysteine sequences. R.Y.T. shares inventors' royalties from these patents. The University of Washington owns a patent on fluorescent GPCR sensors. C.H. and M.J.L. share inventors' royalties from these patents.
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Hoffmann, C., Gaietta, G., Zürn, A. et al. Fluorescent labeling of tetracysteine-tagged proteins in intact cells. Nat Protoc 5, 1666–1677 (2010). https://doi.org/10.1038/nprot.2010.129
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DOI: https://doi.org/10.1038/nprot.2010.129
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