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- Title
Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging.
- Authors
Shi, Xinghua; Jung, Yonil; Lin, Li-Jung; Liu, Cheng; Wu, Cong; Cann, Isaac K O; Ha, Taekjip
- 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.
- Subjects
PROTEIN structure; PROTEIN analysis; DNA polymerases; ALDEHYDES; FLUORIMETRY
- Publication
Nature Methods, 2012, Vol 9, Issue 5, p499
- ISSN
1548-7091
- Publication type
Article
- DOI
10.1038/nmeth.1954