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Two-dimensional gel electrophoresis for identifying proteins that bind DNA or RNA

Nature Protocols volume 2, pages 18391848 (2007) | Download Citation

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Abstract

Electrophoretic mobility shift assays (EMSAs) are commonly used to analyze nucleic acid–protein interactions. When nucleic acid is bound by protein, its mobility during gel electrophoresis is reduced. Similarly, the final position of protein within a complex is shifted when compared to its free state. Here we provide a protocol for a simple approach that uses these mobility differences to identify nucleic acid-binding proteins. Following EMSA, denaturing gel electrophoresis is implemented to provide a second dimension of separation. Protein that binds a specific nucleic acid is identified as a spot(s) whose presence at a particular position(s) is dependent on nucleic acid within the initial binding reaction. The polypeptide in a spot can be subsequently identified by mass spectrometry. As EMSAs can be performed using partially purified or cell extracts, this approach substantially reduces the need for protein purification. It should facilitate the identification of a nucleic acid-binding protein within approximately 4 d.

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Acknowledgements

Ph.D. studentship S02/G031 from the UK Biotechnology and Biological Sciences Research Council (BBSRC). Facilities provided by the BBSRC under the JIF (Joint Infrastructure Fund) Initiative. Helpful comments from colleagues Ian Hope, Simon Baumberg (now deceased) and Peter Stockley.

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  1. Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.

    • Jonathan A Stead
    •  & Kenneth J McDowall

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The authors declare no competing financial interests.

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Correspondence to Kenneth J McDowall.

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DOI

https://doi.org/10.1038/nprot.2007.248

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