Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Single-molecule pull-down for studying protein interactions

Nature Protocols volume 7, pages 445–452 (2012)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Schematic for single-molecule pull-down.
Figure 2: PEG passivation hinders nonspecific protein adsorption.
Figure 3: Preparation of flow chambers.
Figure 4: Determination of lysate dilution factor for PKA tagged with YFP and HA tags.
Figure 5: PKA-AKAP pull-down.

Similar content being viewed by others

References

  1. Alberts, B. The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92, 291–294 (1998).

    Article  CAS  Google Scholar 

  2. Barrios-Rodiles, M. et al. High-throughput mapping of a dynamic signaling network in mammalian cells. Science 307, 1621–1625 (2005).

    Article  CAS  Google Scholar 

  3. Yamada, T. & Bork, P. Evolution of biomolecular networks: lessons from metabolic and protein interactions. Nat. Rev. Mol. Cell Biol. 10, 791–803 (2009).

    Article  CAS  Google Scholar 

  4. Puig, O. et al. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229 (2001).

    Article  CAS  Google Scholar 

  5. Gingras, A.C., Gstaiger, M., Raught, B. & Aebersold, R. Analysis of protein complexes using mass spectrometry. Nat. Rev. Mol. Cell Biol. 8, 645–654 (2007).

    Article  CAS  Google Scholar 

  6. Fields, S. & Song, O. A novel genetic system to detect protein-protein interactions. Nature 340, 245–246 (1989).

    Article  CAS  Google Scholar 

  7. Kerppola, T.K. Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu. Rev. Biophys. 37, 465–487 (2008).

    Article  CAS  Google Scholar 

  8. Reyes-Lamothe, R., Sherratt, D.J. & Leake, M.C. Stoichiometry and architecture of active DNA replication machinery in Escherichia coli. Science 328, 498–501 (2010).

    Article  CAS  Google Scholar 

  9. Li, H., Zhou, D., Browne, H., Balasubramanian, S. & Klenerman, D. Molecule by molecule direct and quantitative counting of antibody-protein complexes in solution. Anal. Chem. 76, 4446–4451 (2004).

    Article  CAS  Google Scholar 

  10. Taguchi, H., Ueno, T., Tadakuma, H., Yoshida, M. & Funatsu, T. Single-molecule observation of protein-protein interactions in the chaperonin system. Nat. Biotechnol. 19, 861–865 (2001).

    Article  CAS  Google Scholar 

  11. Kapanidis, A.N. et al. Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc. Natl. Acad. Sci. USA 101, 8936–8941 (2004).

    Article  CAS  Google Scholar 

  12. Blosser, T.R., Yang, J.G., Stone, M.D., Narlikar, G.J. & Zhuang, X. Dynamics of nucleosome remodelling by individual ACF complexes. Nature 462, 1022–1027 (2009).

    Article  CAS  Google Scholar 

  13. Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011).

    Article  CAS  Google Scholar 

  14. Ulbrich, M.H. & Isacoff, E.Y. Subunit counting in membrane-bound proteins. Nat. Methods 4, 319–321 (2007).

    Article  CAS  Google Scholar 

  15. Hoskins, A.A. et al. Ordered and dynamic assembly of single spliceosomes. Science 331, 1289–1295 (2011).

    Article  CAS  Google Scholar 

  16. Yeom, K.H. et al. Single-molecule approach to immunoprecipitated protein complexes: insights into miRNA uridylation. EMBO Rep. 12, 690–696 (2011).

    Article  CAS  Google Scholar 

  17. Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).

    Article  CAS  Google Scholar 

  18. Fleminger, G., Solomon, B., Wolf, T. & Hadas, E. Effect of polyethylene glycol on the non-specific adsorption of proteins to Eupergit C and agarose. J. Chromatogr. 510, 271–279 (1990).

    Article  CAS  Google Scholar 

  19. Joo, C., Balci, H., Ishitsuka, Y., Buranachai, C. & Ha, T. Advances in single-molecule fluorescence methods for molecular biology. Annu. Rev. Biochem. 77, 51–76 (2008).

    Article  CAS  Google Scholar 

  20. Rothenberg, E. & Ha, T. Single-molecule FRET analysis of helicase functions. Methods Mol. Biol. 587, 29–43 (2010).

    Article  CAS  Google Scholar 

  21. Selvin, P.R. & Ha, T. Single-Molecule Techniques: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2008).

Download references

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

Authors and 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

Authors
  1. Ankur Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ruijie Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yang K Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Taekjip Ha
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

Correspondence to Yang K Xiang or Taekjip Ha.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Immunoprecipitation of endogenous proteins from mouse tissues. (DOC 61 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jain, A., Liu, R., Xiang, Y. et al. Single-molecule pull-down for studying protein interactions. Nat Protoc 7, 445–452 (2012). https://doi.org/10.1038/nprot.2011.452

Download citation

  • Published:

  • Issue Date:

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

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing