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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.

SUBMITTER: Shi X

PROVIDER: S-EPMC3445270 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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

Shi Xinghua X Jung Yonil Y Lin Li-Jung LJ Liu Cheng C Wu Cong C Cann Isaac K O IK Ha Taekjip T

Nature methods 20120401 5

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 e ...[more]

PMID: 22466795

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