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.
© 2021. The Author(s).
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Biosensing Techniques / methods*
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Cell Line
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Crystallography, X-Ray
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Fluorescence
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Fluorescent Dyes / chemistry*
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Humans
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Hydrolases / chemistry*
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Hydrolases / genetics
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Hydrolases / metabolism
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Lysosomal Membrane Proteins / genetics
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Lysosomal Membrane Proteins / metabolism
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Microscopy, Confocal
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Microscopy, Fluorescence / methods
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Mitochondrial Precursor Protein Import Complex Proteins / genetics
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Mitochondrial Precursor Protein Import Complex Proteins / metabolism
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Rhodamines / chemistry
Substances
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Fluorescent Dyes
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LAMP1 protein, human
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Lysosomal Membrane Proteins
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Mitochondrial Precursor Protein Import Complex Proteins
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Recombinant Fusion Proteins
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Rhodamines
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TOMM20 protein, human
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Hydrolases
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haloalkane dehalogenase