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:

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

Nature Protocols volume 2, pages 1839–1848 (2007)Cite this article

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.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

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.

Similar content being viewed by others

References

  1. Fried, M. & Crothers, D.M. Equilibria and kinetics of Lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 9, 6505–6525 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Garner, M.M. & Revzin, A. A gel-electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 9, 3047–3060 (1981).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Fried, M.G. Measurement of protein-DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis 10, 366–376 (1989).

    Article  CAS  PubMed  Google Scholar 

  4. Garner, M.M. & Revzin, A. The use of gel-electrophoresis to detect and study nucleic acid-protein interactions. Trends Biochem. Sci. 11, 395–396 (1986).

    Article  CAS  Google Scholar 

  5. Fried, M.G. & Crothers, D.M. Equilibrium studies of the cyclic-AMP receptor protein-DNA interaction. J. Mol. Biol. 172, 241–262 (1984).

    Article  CAS  PubMed  Google Scholar 

  6. Lohman, T.M., Dehaseth, P.L. & Record, M.T. Pentalysine-deoxyribonucleic acid interactions: a model for the general effects of ion concentrations on the interactions of proteins with nucleic acids. Biochemistry 19, 3522–3530 (1980).

    Article  CAS  PubMed  Google Scholar 

  7. Lohman, T.M., DeHaseth, P.L. & Record, M.T. Analysis of ion concentration effects on kinetics of protein-nucleic acid interactions. Application to Lac repressor-operator interactions. Biophys. Chem. 8, 281–294 (1978).

    Article  CAS  PubMed  Google Scholar 

  8. Fried, M.G. & Crothers, D.M. Kinetics and mechanism in the reaction of gene regulatory proteins with DNA. J. Mol. Biol. 172, 263–282 (1984).

    Article  CAS  PubMed  Google Scholar 

  9. Gadgil, H., Jurado, L.A. & Jarrett, H.W. DNA affinity chromatography of transcription factors. Anal. Biochem. 290, 147–178 (2001).

    Article  CAS  PubMed  Google Scholar 

  10. Moxley, R.A., Jarrett, H.W. & Mitra, S. Methods for transcription factor separation. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 797, 269–288 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Pinolroma, S., Choi, Y.D. & Dreyfuss, G. Immunological methods for purification and characterization of heterogeneous nuclear ribonucleoprotein-particles. Methods Enzymol. 181, 317–325 (1990).

    Article  CAS  Google Scholar 

  12. Nordhoff, E. et al. Rapid identification of DNA-binding proteins by mass spectrometry. Nat. Biotechnol. 17, 884–888 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Yaneva, M. & Tempst, P. Affinity capture of specific DNA-binding proteins for mass spectrometric identification. Anal. Chem. 75, 6437–6448 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Woo, A.J., Dods, J.S., Susanto, E., Ulgiati, D. & Abraham, L.J. A proteomics approach for the identification of DNA binding activities observed in the electrophoretic mobility shift assay. Mol. Cell. Proteomics 1, 472–478 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Stead, J.A., Keen, J.N. & McDowall, K.J. The identification of nucleic acid-interacting proteins using a simple proteomics-based approach that directly incorporates the electrophoretic mobility shift assay. Mol. Cell. Proteomics 5, 1697–1702 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Hope, I.A. & Struhl, K. Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast. Cell 46, 885–894 (1986).

    Article  CAS  PubMed  Google Scholar 

  17. Bremer, H. & Dennis, P. Modulation of chemical composition and other parameters of the cell by growth rate. in Escherichia coli and Salmonella: Cellular and Molecular Biology (ed. Neidhardt, F.C.) 1553–1569 (ASM Press, Washington D.C., 1996).

    Google Scholar 

  18. Sherman, F. Getting started with yeast. in Guide to Yeast Genetics and Molecular and Cell Biology, Part B (eds. Guthrie, C. and Fink, G.R.) 3–41 (Academic Press, San Diego, 2002).

    Chapter  Google Scholar 

  19. Jackson, S.P. & Tjian, R. Purification and analysis of RNA polymerase II transcription factors by using wheat germ-agglutinin affinity chromatography. Proc. Natl. Acad. Sci. USA 86, 1781–1785 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Uguru, G.C. et al. Transcriptional activation of the pathway-specific regulator of the actinorhodin biosynthetic genes in Streptomyces coelicolor. Mol. Microbiol. 58, 131–150 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Allen, S.V. & Miller, E.S. RNA-binding properties of in vitro expressed histidine-tagged RB69 RegA translational repressor protein. Anal. Biochem. 269, 32–37 (1999).

    Article  CAS  PubMed  Google Scholar 

  23. Callaghan, A.J. et al. Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover. Nature 437, 1187–1191 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Jiao, X., Trifillis, P. & Kiledjian, M. Identification of target messenger RNA substrates for the murine deleted in azoospermia-like RNA-binding protein. Biol. Reprod. 66, 475–485 (2002).

    Article  CAS  PubMed  Google Scholar 

  25. Kaberdin, V.R., Chao, Y.H. & Sue, L.C. RNase E cleaves at multiple sites in bubble regions of RNA I stem loops yielding products that dissociate differentially from the enzyme. J. Biol. Chem. 271, 13103–13109 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. Sengupta, T.K., Gordon, J. & Spicer, E.K. RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities. Nucleic Acids Res. 29, 1175–1184 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Ta, M. & Vrati, S. Mov34 protein from mouse brain interacts with the 3 noncoding region of Japanese encephalitis virus. J. Virol. 74, 5108–5115 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Grunwald, M.E., Yu, W.P., Yu, H.H. & Yau, K.W. Identification of a domain on the beta-subunit of the rod cGMP-gated cation channel that mediates inhibition by calcium-calmodulin. J. Biol. Chem. 273, 9148–9157 (1998).

    Article  CAS  PubMed  Google Scholar 

  29. Dangerfield, J.A., Windbichler, N., Salmons, B., Gunzburg, W.H. & Schroder, R. Enhancement of the StreptoTag method for isolation of endogenously expressed proteins with complex RNA binding targets. Electrophoresis 27, 1874–1877 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Bachler, M., Schroeder, R. & von Ahsen, U. StreptoTag: a novel method for the isolation of RNA-binding proteins. RNA 5, 1509–1516 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Moxley, R.A. & Jarrett, H.W. Oligonucleotide trapping method for transcription factor purification systematic optimization using electrophoretic mobility shift assay. J. Chromatogr. A 1070, 23–34 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Gadgil, H. & Jarrett, H.W. Oligonucleotide trapping method for purification of transcription factors. J. Chromatogr. A 966, 99–110 (2002).

    Article  CAS  PubMed  Google Scholar 

  33. Buratowski, S. & Chodosh, L.A. Mobility shift DNA-binding assay using gel electrophoresis. in Current Protocols in Molecular Biology (eds. Ausubel, F.M. et al.) 12.12.11–12.12.11 (John Wiley and Sons, New York, 1996).

    Google Scholar 

  34. Lane, D., Prentki, P. & Chandler, M. Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol. Rev. 56, 509–528 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Fried, M.G. & Garner, M.M. The electrophoretic mobility shift assay (EMSA) for detection and analysis of protein-DNA interactions. in Molecular Biology Methods and Applications (ed. Tietz, D.) 239–271 (Elsevier, New York, 1998).

    Google Scholar 

  36. Carey, J. Gel retardation. Methods Enzymol. 208, 103–117 (1991).

    Article  CAS  PubMed  Google Scholar 

  37. Hellman, L.M. & Fried, M. Electrophoretic mobility shift assay (EMSA) for protein-nucleic acid interaction. Nat. Protoc. 2, 1849–1861 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Li, Y., Jiang, Z.Z., Chen, H.X. & Ma, W.J. A modified quantitative EMSA and its application in the study of RNA-protein interactions. J. Biochem. Biophys. Methods 60, 85–96 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Kang, J., Lee, M.S. & Gorenstein, D.G. Quantitative analysis of chemiluminescence signals using a cooled charge-coupled device camera. Anal. Biochem. 345, 66–71 (2005).

    Article  CAS  PubMed  Google Scholar 

  40. Forwood, J.K. & Jans, D.A. Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging. Electrophoresis 27, 3166–3170 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Jing, D., Agnew, J., Patton, W.F., Hendrickson, J. & Beechem, J.M. A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels. Proteomics 3, 1172–1180 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Vossen, K.M., Wolz, R., Daugherty, M.A. & Fried, M.G. Role of macromolecular hydration in the binding of the Escherichia coli cyclic AMP receptor to DNA. Biochemistry 36, 11640–11647 (1997).

    Article  CAS  PubMed  Google Scholar 

  43. Garner, M.M. & Rau, D.C. Water release associated with specific binding of gal repressor. EMBO J. 14, 1257–1263 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Carrette, O., Burkhard, P., Sanchez, J.-C. & Hochstrasser, D. State-of-the-art two-dimensional gel electrophoresis: a key tool of proteomics research. Nat. Protoc. 1, 812–823 (2006).

    Article  CAS  PubMed  Google Scholar 

  46. Chevallet, M., Luche, S. & Rabilloud, T. Silver staining of proteins in polyacrylamide gels. Nat. Protoc. 1, 1852–1858 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Havlis, J., Thomas, H., Sebela, M. & Shevchenko, A. Fast-response proteomics by accelerated in-gel digestion of proteins. Anal. Chem. 75, 1300–1306 (2003).

    Article  CAS  PubMed  Google Scholar 

  48. Kaberdin, V.R. & McDowall, K.J. Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix. Genome Res. 13, 1961–1965 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

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

    Jonathan A Stead & Kenneth J McDowall

Authors
  1. Jonathan A Stead
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kenneth J McDowall
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kenneth J McDowall.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

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

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

Advanced 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