Analysis of DNA-Protein Interactions Using PAGE: Band-Shift Assays

  • Lynn Powell
Part of the Methods in Molecular Biology book series (MIMB, volume 1054)


The band-shift assay using polyacrylamide gel electrophoresis is a powerful technique used to investigate DNA–protein interactions. The basis of the method is the separation of free DNA from DNA–protein complexes by virtue of differences in charge, size, and shape. The band-shift assay can be used to determine thermodynamic and kinetic binding constants and also to analyze the composition and stoichiometries of DNA–protein complexes.

Key words

Band shift DNA–protein interaction DNA binding constant EMSA PAGE Gel retardation 



I would like to acknowledge the help of Professors Noreen Murray and Andrew Jarman in whose laboratories this work took place. Thanks are also due to my colleagues in the Murray and Jarman laboratories, in particular David Dryden, Laurie Cooper, and Petra zur Lage for their help and advice with protein purification.


  1. 1.
    Garner MM, Revzin A (1981) 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 9:3047–3060PubMedCrossRefGoogle Scholar
  2. 2.
    Fried M, Crothers DM (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res 9:6505–6525PubMedCrossRefGoogle Scholar
  3. 3.
    Powell LM, Dryden DT, Willcock DF, Pain RH, Murray NE (1993) DNA recognition by the EcoK methyltransferase. The influence of DNA methylation and the cofactor S-adenosyl-l-methionine. J Mol Biol 234:60–71PubMedCrossRefGoogle Scholar
  4. 4.
    Powell LM, Zur Lage PI, Prentice DR, Senthinathan B, Jarman AP (2004) The proneural proteins Atonal and Scute regulate neural target genes through different E-box binding sites. Mol Cell Biol 24:9517–9526PubMedCrossRefGoogle Scholar
  5. 5.
    Dey B, Thukral S, Krishnan S, Chakrobarty M, Gupta S, Manghani C, Rani V (2012) DNA–protein interactions: methods for detection and analysis. Mol Cell Biochem 365:279–299PubMedCrossRefGoogle Scholar
  6. 6.
    Henriksson-Peltola P, Sehlen W, Haggard-Ljungquist E (2007) Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis. Nucleic Acids Res 35:3181–3191PubMedCrossRefGoogle Scholar
  7. 7.
    Buratowski S, Chodosh LA (2001) Mobility shift DNA-binding assay using gel electrophoresis. In: Ausubel FM et al (eds) Current protocols in molecular biology, 36: 12.2.1–12.2.11, John Wiley and Sons inc.Google Scholar
  8. 8.
    Lane D, Prentki P, Chandler M (1992) Use of gel retardation to analyze protein–nucleic acid interactions. Microbiol Rev 56:509–528PubMedGoogle Scholar
  9. 9.
    Jiang D, Jarrett HW, Haskins WE (2009) Methods for proteomic analysis of transcription factors. J Chromatogr 1216:6881–6889CrossRefGoogle Scholar
  10. 10.
    zur Lage PI, Powell LM, Prentice DR, McLaughlin P, Jarman AP (2004) EGF receptor signaling triggers recruitment of Drosophila sense organ precursors by stimulating proneural gene autoregulation. Dev Cell 7:687–696PubMedCrossRefGoogle Scholar
  11. 11.
    Powell LM, Murray NE (1995) S-adenosyl methionine alters the DNA contacts of the EcoKI methyltransferase. Nucleic Acids Res 23:967–974PubMedCrossRefGoogle Scholar
  12. 12.
    Powell LM, Dryden DT, Murray NE (1998) Sequence-specific DNA binding by EcoKI, a type IA DNA restriction enzyme. J Mol Biol 283:963–976PubMedCrossRefGoogle Scholar
  13. 13.
    Hellman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions. Nat Protoc 2:1849–1861PubMedCrossRefGoogle Scholar
  14. 14.
    Kothinti R, Tabatabai NM, Petering DH (2011) Electrophoretic mobility shift assay of zinc finger proteins: competition for Zn(2+) bound to Sp1 in protocols including EDTA. J Inorg Biochem 105:569–576PubMedCrossRefGoogle Scholar
  15. 15.
    Chen J, Villanueva N, Rould MA, Morrical SW (2010) Insights into the mechanism of Rad51 recombinase from the structure and properties of a filament interface mutant. Nucleic Acids Res 38:4889–4906PubMedCrossRefGoogle Scholar
  16. 16.
    Carey MF, Peterson CL, Smale ST (2012) Experimental strategies for the identification of DNA-binding proteins. Cold Spring Harb Protoc 2012:18–33PubMedGoogle Scholar
  17. 17.
    Cutler PE (2004) Protein purification protocols. Methods Mol Biol (Clifton, NJ) 244: 1–496Google Scholar
  18. 18.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harb Laboratory Press, NewYorkGoogle Scholar
  19. 19.
    Kurien BT, Scofield RH (2009) Introduction to protein blotting. Methods Mol Biol (Clifton, NJ) 536:9–22CrossRefGoogle Scholar
  20. 20.
    Forwood JK, Jans DA (2006) Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging. Electrophoresis 27:3166–3170PubMedCrossRefGoogle Scholar
  21. 21.
    Pagano JM, Clingman CC, Ryder SP (2011) Quantitative approaches to monitor protein–nucleic acid interactions using fluorescent probes. RNA 17:14–20PubMedCrossRefGoogle Scholar
  22. 22.
    Revzin A (1989) Gel electrophoresis assays for DNA–protein interactions. Biotechniques 7:346–355PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2013

Authors and Affiliations

  • Lynn Powell
    • 1
  1. 1.Centre for Integrative Physiology, School of Biomedical SciencesUniversity of EdinburghEdinburghUK

Personalised recommendations