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:

Immunoprecipitation of mRNA-protein complexes

Abstract

Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)–RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: (ac) Protein lysate, a, is incubated with protein A–agarose beads, b, to preclear any nonspecific binding of the lysate to the beads, c.

Similar content being viewed by others

References

  1. Zielinski, J. et al. In vivo identification of ribonucleoprotein-RNA interactions. Proc. Natl. Acad. Sci. USA 103, 1557–1562 (2006).

    Article  CAS  Google Scholar 

  2. Zeng, F. et al. A protocol for PAIR: PNA-assisted identification of RNA-binding proteins in living cells. Nat. Protocols (2006) (doi:10.1038/nprot.2006.81).

  3. Tenenbaum, S.A., Carson, C.C., Lager, P.J. & Keene, J.D. Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc. Natl. Acad. Sci. USA 97, 14085–14090 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Maronski for help with culturing the neurons. This work was funded by grants AG9900 and MH58561 from the NIH to JE and the Swedish Science Foundation Grants Med and NT, and European Community Grant QLRT-2001-01989 to U.L.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to James Eberwine.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Peritz, T., Zeng, F., Kannanayakal, T. et al. Immunoprecipitation of mRNA-protein complexes. Nat Protoc 1, 577–580 (2006). https://doi.org/10.1038/nprot.2006.82

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2006.82

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

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