Immunoprecipitation of mRNA-protein complexes (original) (raw)

Nature Protocols volume 1, pages 577–580 (2006)Cite this article

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.

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

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References

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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.

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Authors and Affiliations

  1. Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, 19104, Pennsylvania, USA
    Tiina Peritz, Fanyi Zeng, Theresa J Kannanayakal & James Eberwine
  2. Shanghai Institute of Medical Genetics, Shanghai Jiao Tong University, Shanghai, 200040, China
    Fanyi Zeng
  3. Department of Neurochemistry, Stockholm University, SE-106 91, Stockholm, Sweden
    Kalle Kilk, Emelía Eiríksdóttir & Ulo Langel

Authors

  1. Tiina Peritz
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  2. Fanyi Zeng
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  3. Theresa J Kannanayakal
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  4. Kalle Kilk
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  5. Emelía Eiríksdóttir
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  6. Ulo Langel
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  7. James Eberwine
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Corresponding author

Correspondence toJames Eberwine.

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The authors declare no competing financial interests.

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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

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