Skip to main content
SpringerLink
Log in

Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK)

  • Brief Communication
  • Published:

From Nature Methods

View current issue Submit your manuscript

Abstract

Present screening methods for protein-protein interactions (PPIs) rely on the overexpression of artificial fusion proteins, making it difficult to assess in vivo relevance. Here we combine stable isotope labeling with amino acids in cell culture (SILAC), RNA interference (RNAi), coimmunoprecipitation and quantitative mass-spectrometry analysis to detect cellular interaction partners of endogenous proteins in mammalian cells with very high confidence. We used this screen to identify interaction partners of β-catenin and Cbl.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Strategy to screen for endogenous PPIs.
Figure 2: Identification of endogenous β-catenin interaction partners.
Figure 3: QUICK screen for interaction partners of Cbl.

Similar content being viewed by others

References

  1. Bader, J.S., Chaudhuri, A., Rothberg, J.M. & Chant, J. Nat. Biotechnol. 22, 78–85 (2004).

    Article  CAS  Google Scholar 

  2. Li, S. et al. Science 303, 540–543 (2004).

    Article  CAS  Google Scholar 

  3. Zhu, H., Bilgin, M. & Snyder, M. Annu. Rev. Biochem. 72, 783–812 (2003).

    Article  CAS  Google Scholar 

  4. Aebersold, R. & Mann, M. Nature 422, 198–207 (2003).

    Article  CAS  Google Scholar 

  5. Ong, S.E. & Mann, M. Nat. Chem. Biol. 1, 252–262 (2005).

    Article  CAS  Google Scholar 

  6. Ong, S.E. et al. Mol. Cell. Proteomics 1, 376–386 (2002).

    Article  CAS  Google Scholar 

  7. Schulze, W.X. & Mann, M. J. Biol. Chem. 279, 10756–10764 (2004).

    Article  CAS  Google Scholar 

  8. Gunsalus, K.C. & Piano, F. Curr. Opin. Cell Biol. 17, 3–8 (2005).

    Article  CAS  Google Scholar 

  9. Reya, T. & Clevers, H. Nature 434, 843–850 (2005).

    Article  CAS  Google Scholar 

  10. van de Wetering, M. et al. EMBO Rep. 4, 609–615 (2003).

    Article  CAS  Google Scholar 

  11. Kucerova, D., Sloncova, E., Tuhackova, Z., Vojtechova, M. & Sovova, V. Int. J. Mol. Med. 8, 695–698 (2001).

    CAS  Google Scholar 

  12. Kim, I.J. et al. Clin. Cancer Res. 9, 2920–2925 (2003).

    CAS  Google Scholar 

  13. Schmidt, M.H. & Dikic, I. Nat. Rev. Mol. Cell Biol. 6, 907–919 (2005).

    Article  CAS  Google Scholar 

  14. Silva, J.M. et al. Nat. Genet. 37, 1281–1288 (2005).

    Article  CAS  Google Scholar 

  15. Uhlen, M. et al. Mol. Cell. Proteomics 4, 1920–1932 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank B. Blagoev from the Center of Experimental Bioinformatics for fruitful discussions, J.V. Olsen and other members of our department for discussion and assistance, H. Clevers for generously providing us with the inducible β-catenin knockdown cell line and the Interaction Proteome project of the European Union for funding.

Author information

Authors and Affiliations

  1. Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsried, D-82152, Germany

    Matthias Selbach & Matthias Mann

Authors
  1. Matthias Selbach
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Matthias Mann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matthias Mann.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Sequences and normalized abundance ratios of the quantified peptides for β-catenin and three detected interaction partners. (DOC 38 kb)

Supplementary Table 2

Sequences and normalized abundance ratios of the quantified peptides for Cbl and four detected interaction partners. (DOC 39 kb)

Supplementary Table 3

Additional proteins identified with increased abundance ratios in the Cbl pulldown. (DOC 40 kb)

Supplementary Table 4

Ratio of proteins identified in the whole cell lysates after Cbl knock-down. (DOC 201 kb)

Supplementary Methods (DOC 50 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Selbach, M., Mann, M. Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nat Methods 3, 981–983 (2006). https://doi.org/10.1038/nmeth972

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth972

  • Springer Nature America, Inc.

This article is cited by

Search

Navigation