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 journalArticlepeer-review

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 languageEnglish (US)
Pages (from-to)499-503
Number of pages5
JournalNature Methods
Volume9
Issue number5
DOIs
StatePublished - May 2012
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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