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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aldehydes / chemistry*
  • Carbocyanines / chemical synthesis*
  • Carbocyanines / chemistry
  • DNA-Directed DNA Polymerase / chemistry*
  • Fluorescent Dyes / chemical synthesis*
  • Fluorescent Dyes / chemistry
  • Kinetics
  • Microscopy, Fluorescence


  • Aldehydes
  • Carbocyanines
  • Fluorescent Dyes
  • DNA-Directed DNA Polymerase