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Engineered HaloTag variants for fluorescence lifetime multiplexing

MPS-Authors
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Frei,  Michelle S.
Chemical Biology, Max Planck Institute for Medical Research, Max Planck Society;

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Tarnawski,  Miroslaw
Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Max Planck Society;

/persons/resource/persons243203

Koch,  Birgit
Chemical Biology, Max Planck Institute for Medical Research, Max Planck Society;

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Hiblot,  Julien
Chemical Biology, Max Planck Institute for Medical Research, Max Planck Society;

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Johnsson,  Kai
Chemical Biology, Max Planck Institute for Medical Research, Max Planck Society;

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Citation

Frei, M. S., Tarnawski, M., Roberti, M. J., Koch, B., Hiblot, J., & Johnsson, K. (2022). Engineered HaloTag variants for fluorescence lifetime multiplexing. Nature Methods, 19, 65-70. doi:10.1038/s41592-021-01341-x.


Cite as: http://hdl.handle.net/21.11116/0000-0009-A743-F
Abstract
Self-labeling protein tags such as HaloTag are powerful tools that can label fusion proteins with synthetic fluorophores for use in fluorescence microscopy. Here we introduce HaloTag variants with either increased or decreased brightness and fluorescence lifetime compared with HaloTag7 when labeled with rhodamines. Combining these HaloTag variants enabled live-cell fluorescence lifetime multiplexing of three cellular targets in one spectral channel using a single fluorophore and the generation of a fluorescence lifetime-based biosensor. Additionally, the brightest HaloTag variant showed up to 40% higher brightness in live-cell imaging applications.