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

• DOI: 10.1038/nmeth.1954
• Publication Date: 2012-04-01T17:59:28Z
• Authors: Xinghua Shi; Yonil Jung; Li-Jung Lin; Cheng Liu; Cong Wu; Isaac K O Cann; Taekjip Ha
• Keywords: Aldehydes; Kinetics; 0303 health sciences; 03 medical and health sciences; Microscopy, Fluorescence; Biophysics; DNA-Directed DNA Polymerase; Carbocyanines; Article; Fluorescent Dyes

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.