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

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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Figure 1: Preparation of a tube gel.
Figure 2: Preparation of a resolving gel.
Figure 3: Photograph of the core of an electrophoresis cell with a tube holder and tube gels attached.
Figure 4: Extrusion of a tube gel.
Figure 5: Attachment of the tube gel to the SDS-polyacrylamide slab gel.
Figure 6: Identifying the migration path in the second dimension of proteins that bound the probe.
Figure 7: Anticipated results.

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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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Correspondence to Kenneth J McDowall.

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Stead, J., McDowall, K. Two-dimensional gel electrophoresis for identifying proteins that bind DNA or RNA. Nat Protoc 2, 1839–1848 (2007). https://doi.org/10.1038/nprot.2007.248

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