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

The combination of a genetically encoded aldehyde tag and optimized labeling method allows high-efficiency, site-specific labeling of tagged proteins after purification or in cell extracts. The authors use the high labeling efficiency for single-molecule measurements of the dynamic interactions between two DNA polymerases and polymerase processivity factor bound to DNA. 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.