Protocol | Published:

Single-molecule pull-down for studying protein interactions

Nature Protocols volume 7, pages 445452 (2012) | Download Citation

Abstract

This protocol describes a single-molecule pull-down (SiMPull) assay for analyzing physiological protein complexes. The assay combines the conventional pull-down assay with single-molecule total internal reflection fluorescence (TIRF) microscopy and allows the probing of single macromolecular complexes directly from cell or tissue extracts. In this method, antibodies against the protein of interest are immobilized on a passivated microscope slide. When cell extracts are applied, the surface-tethered antibody captures the protein together with its physiological interaction partners. After washing away the unbound components, single-molecule fluorescence microscopy is used to probe the pulled-down proteins. Captured proteins are visualized through genetically encoded fluorescent protein tags or through antibody labeling. Compared with western blot analysis, this ultrasensitive assay requires considerably less time and reagents and provides quantitative data. Furthermore, SiMPull can distinguish between multiple association states of the same protein. SiMPull is generally applicable to proteins from a variety of cellular contexts and to endogenous proteins. Starting with the cell extracts and passivated slides, the assay requires 1.5–2.5 h for data acquisition and analysis.

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Acknowledgements

We thank B. Ramani, Y. Ishitsuka and K. Ragunathan for help with developing the protocol. This work was funded by US National Institutes of Health grants (AI083025, GM065367 to T.H.; HL082846 to Y.K.X.). Additional support was provided by National Science Foundation grants (0646550, 0822613 to T.H.). T.H. is an investigator with the Howard Hughes Medical Institute.

Author information

Affiliations

  1. Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Ankur Jain
    •  & Taekjip Ha
  2. Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Ankur Jain
    • , Yang K Xiang
    •  & Taekjip Ha
  3. Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Ruijie Liu
    •  & Yang K Xiang
  4. Howard Hughes Medical Institute, Urbana, Illinois, USA.

    • Taekjip Ha

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Contributions

A.J., Y.K.X. and T.H. designed the research. R.L. prepared the cell extracts. A.J. conducted single-molecule experiments and analyzed the data. A.J., Y.K.X. and T.H. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Yang K Xiang or Taekjip Ha.

Supplementary information

Word documents

  1. 1.

    Supplementary Methods

    Immunoprecipitation of endogenous proteins from mouse tissues.

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DOI

https://doi.org/10.1038/nprot.2011.452

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