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Nat Methods. 2012 Apr 1;9(5):499-503. doi: 10.1038/nmeth.1954.

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

Author information

1
Howard Hughes Medical Institute, Urbana, Illinois, USA.

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.

PMID:
22466795
PMCID:
PMC3445270
DOI:
10.1038/nmeth.1954
[Indexed for MEDLINE]
Free PMC Article

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