Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging

Xinghua Shi; Yonil Jung; Li Jung Lin; Cheng Liu; Cong Wu; Isaac K.O. Cann; Taekjip Ha

doi:10.1038/nmeth.1954

Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging

Xinghua Shi, Yonil Jung, Li Jung Lin, Cheng Liu, Cong Wu, Isaac K.O. Cann, Taekjip Ha

Research output: Contribution to journal › Article › peer-review

52 Scopus citations

Abstract

A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescence labeling with high efficiency, specificity and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ∼100% efficiency while maintaining the biological function. We show that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We also show the unique power of our method in single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor.

Original language	English (US)
Pages (from-to)	499-503
Number of pages	5
Journal	Nature Methods
Volume	9
Issue number	5
DOIs	https://doi.org/10.1038/nmeth.1954
State	Published - May 2012
Externally published	Yes

ASJC Scopus subject areas

Biotechnology
Biochemistry
Molecular Biology
Cell Biology

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10.1038/nmeth.1954

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title = "Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging",

abstract = "A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescence labeling with high efficiency, specificity and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ∼100% efficiency while maintaining the biological function. We show that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We also show the unique power of our method in single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor.",

author = "Xinghua Shi and Yonil Jung and Lin, {Li Jung} and Cheng Liu and Cong Wu and Cann, {Isaac K.O.} and Taekjip Ha",

note = "Funding Information: We thank C. Bertozzi (University of California, Berkeley) for providing the plasmid DNA for FGE through Addgene, J. Fei for the suggestion of using hydrophobic interaction chromatography, and K. Ragunathan for a critical reading of the manuscript. This work was supported by the US National Institutes of Health grants GM065367 and AI083025 (to T.H.) and the US National Science Foundation grants MCB-0238451 (to I.C.) and PHY-0822613 (to T.H.).",

year = "2012",

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doi = "10.1038/nmeth.1954",

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AU - Shi, Xinghua

AU - Jung, Yonil

AU - Lin, Li Jung

AU - Liu, Cheng

AU - Wu, Cong

AU - Cann, Isaac K.O.

AU - Ha, Taekjip

N1 - Funding Information: We thank C. Bertozzi (University of California, Berkeley) for providing the plasmid DNA for FGE through Addgene, J. Fei for the suggestion of using hydrophobic interaction chromatography, and K. Ragunathan for a critical reading of the manuscript. This work was supported by the US National Institutes of Health grants GM065367 and AI083025 (to T.H.) and the US National Science Foundation grants MCB-0238451 (to I.C.) and PHY-0822613 (to T.H.).

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N2 - A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescence labeling with high efficiency, specificity and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ∼100% efficiency while maintaining the biological function. We show that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We also show the unique power of our method in single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor.

AB - A major hurdle for molecular mechanistic studies of many proteins is the lack of a general method for fluorescence labeling with high efficiency, specificity and speed. By incorporating an aldehyde motif genetically into a protein and improving the labeling kinetics substantially under mild conditions, we achieved fast, site-specific labeling of a protein with ∼100% efficiency while maintaining the biological function. We show that an aldehyde-tagged protein can be specifically labeled in cell extracts without protein purification and then can be used in single-molecule pull-down analysis. We also show the unique power of our method in single-molecule studies on the transient interactions and switching between two quantitatively labeled DNA polymerases on their processivity factor.

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Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging

Abstract

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