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

Nature Methods volume 9, pages 499–503 (2012)Cite this article

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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.

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Figure 1: Development and validation of the quantitative fluorescence labeling of an aldehyde-tagged protein.
Figure 2: General applicability of labeling an aldehyde-tagged protein.
Figure 3: Specific and efficient labeling of unpurified Ald6N-DinB in cell extracts and single-molecule pull-down.
Figure 4: Real-time observation of the transient interactions and switching between PolBI and DinB.

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Acknowledgements

We thank C. Bertozzi (University of California, Berkeley) for providing the plasmid DNA for FGE through Addgene, J. Fei for the suggestion of using hydrophobic interaction chromatography, and K. Ragunathan for a critical reading of the manuscript. This work was supported by the US National Institutes of Health grants GM065367 and AI083025 (to T.H.) and the US National Science Foundation grants MCB-0238451 (to I.C.) and PHY-0822613 (to T.H.).

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Authors and Affiliations

  1. Howard Hughes Medical Institute, Urbana, Illinois, USA

    Xinghua Shi & Taekjip Ha

  2. Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Xinghua Shi, Isaac K O Cann & Taekjip Ha

  3. Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Xinghua Shi, Cheng Liu & Taekjip Ha

  4. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Yonil Jung & Cong Wu

  5. Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Li-Jung Lin & Isaac K O Cann

  6. Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA

    Isaac K O Cann

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  1. Xinghua Shi
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Contributions

X.S. and T.H. conceived the study; X.S. designed the experiments; X.S., Y.J., L.-J.L., C.L. and C.W. performed the experiments and analyzed the data; X.S., I.K.O.C. and T.H. wrote the manuscript.

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Correspondence to Taekjip Ha.

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Supplementary Figures 1–7, Supplementary Table 1, Supplementary Notes 1–6 (PDF 1020 kb)

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Shi, X., Jung, Y., Lin, LJ. et al. Quantitative fluorescence labeling of aldehyde-tagged proteins for single-molecule imaging. Nat Methods 9, 499–503 (2012). https://doi.org/10.1038/nmeth.1954

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