Isolation of specific RNA-binding proteins using the streptomycin-binding RNA aptamer (original) (raw)

Nature Protocols volume 1, pages 637–640 (2006)Cite this article

Abstract

Here we report a simple and cheap one-step affinity purification protocol for isolating RNAs or proteins that interact with selected functional RNAs. The streptomycin-binding aptamer, termed 'StreptoTag,' is embedded in or fused to either end of any RNA of interest. The resulting hybrid RNA can then be immobilized on a streptomycin affinity matrix. When a complex protein mixture or total cellular lysate is applied to the matrix, subsequent elution with free streptomycin allows efficient recovery of specific ribonucleoprotein or RNA-RNA complexes. The method was successfully used to purify yeast and phage RNA-binding proteins and group II intron, viral and bacterial noncoding RNA (ncRNA)-binding proteins. The selective enrichment of bacterial mRNAs that bind ncRNAs has also been demonstrated. Once the affinity matrix, the RNA construct and the protein extracts have been prepared, the experimental procedure can be performed in 1–2 h.

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Figure 1: StreptoTag affinity purification procedure.

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Acknowledgements

Work in our laboratory is funded by the Austrian Science fund projects Z-72 and F1703, by the European Community BACRNA FP6-018618 and by the Austrian BMBWK Gen AU programme.

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Authors and Affiliations

  1. Department of Biological Sciences, Imperial College London, Imperial College Road, London, SW7 2AZ, UK
    Nikolai Windbichler
  2. Max F. Perutz Laboratories, University of Vienna, Dr. Bohrgasse 9/5, Vienna, A-1030, Austria
    Renée Schroeder

Authors

  1. Nikolai Windbichler
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  2. Renée Schroeder
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Correspondence toRenée Schroeder.

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The authors declare no competing financial interests.

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Windbichler, N., Schroeder, R. Isolation of specific RNA-binding proteins using the streptomycin-binding RNA aptamer.Nat Protoc 1, 637–640 (2006). https://doi.org/10.1038/nprot.2006.95

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