title: Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging.
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.
authors.affiliation: Howard Hughes Medical Institute, Urbana, Illinois, USA.
authors.forename: Xinghua
authors.initials: X
authors.lastname: Shi
authors.forename: Yonil
authors.initials: Y
authors.lastname: Jung
authors.forename: Li-Jung
authors.initials: LJ
authors.lastname: Lin
authors.forename: Cheng
authors.initials: C
authors.lastname: Liu
authors.forename: Cong
authors.initials: C
authors.lastname: Wu
authors.forename: Isaac K O
authors.initials: IK
authors.lastname: Cann
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D002232:Carbocyanines / Q000138:chemical synthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence
chemical_list: D000447:Aldehydes; D002232:Carbocyanines; D005456:Fluorescent Dyes; D004259:DNA-Directed DNA Polymerase