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Hox Genes pp 211-230 | Cite as

Measuring Hox-DNA Binding by Electrophoretic Mobility Shift Analysis

  • Kelly Churion
  • Ying Liu
  • Hao-Ching Hsiao
  • Kathleen S. Matthews
  • Sarah E. BondosEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1196)

Abstract

Understanding gene regulation by Hox transcription factors requires understanding the forces that underlie DNA binding by these proteins. Electrophoretic mobility shift analysis (EMSA) not only allows measurement of protein affinity and cooperativity but also permits visualization of differently migrating protein-DNA complexes, including complexes with different compositions or complexes with identical compositions yet assembled in different geometries. Furthermore, protein activity can be measured, allowing correction of binding constants for the percentage of protein that is properly folded and capable of binding DNA. Protocols for measuring protein activity and the equilibrium DNA-binding dissociation constant (K d) are provided. This versatile assay system can be adjusted based on specific needs to measure other parameters, including the kinetic association and dissociation constants (k a and k d) and the formation of heterologous protein-protein interactions.

Key words

Hox Gel retardation Gel shift Electrophoretic mobility shift analysis EMSA DNA binding Activity Affinity Cooperativity 

Notes

Acknowledgements

This work was supported by an RDEAP grant from the Texas A&M Health Science Center to S.E.B. and a Robert A. Welch Foundation grant (C-576) to K.S.M.

References

  1. 1.
    Carey J (1991) Gel retardation. Methods Enzymol 208:103–117PubMedCrossRefGoogle Scholar
  2. 2.
    Senear DF, Brenowitz M (1991) Determination of binding constants for cooperative site-specific protein-DNA interactions using the gel mobility-shift assay. J Biol Chem 266:13661–13671PubMedGoogle Scholar
  3. 3.
    Liu Y, Matthews KS, Bondos SE (2009) Internal regulatory interactions determine DNA binding specificity by a Hox transcription factor. J Mol Biol 390:760–774PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Beachy PA, Varkey J, Young KE et al (1993) Cooperative binding of an Ultrabithorax homeodomain protein to nearby and distant DNA sites. Mol Cell Biol 13:6941–6956PubMedCentralPubMedGoogle Scholar
  5. 5.
    Joshi R, Sun L, Mann R (2010) Dissecting the functional specificities of two Hox proteins. Genes Dev 24:1533–1545PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Bondos SE, Bicknell AA (2003) Detection and prevention of protein aggregation before, during, and after purification. Anal Biochem 316:223–231PubMedCrossRefGoogle Scholar
  7. 7.
    Lane D, Prentki P, Chandler M (1992) Use of gel retardation to analyze protein-nucleic acid interactions. Microbiol Rev 56:509–528PubMedCentralPubMedGoogle Scholar
  8. 8.
    Hellman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc 2:1849–1861PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    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 9:6505–6525CrossRefGoogle Scholar
  10. 10.
    Gerstle JT, Fried MG (1991) Measurement of binding-kinetics using the gel-electrophoresis mobility shift assay. Electrophoresis 14:725–731CrossRefGoogle Scholar
  11. 11.
    Bondos SE, Catanese DJ Jr, Tan XX et al (2004) Hox transcription factor Ultrabithorax physically and genetically interacts with Disconnected Interacting Protein 1, a double-stranded RNA-binding protein. J Biol Chem 279:26433–26444PubMedCrossRefGoogle Scholar
  12. 12.
    Merabet S, Saadaoui M, Sambrani N et al (2007) A unique Extradenticle recruitment mode in the Drosophila Hox protein Ultrabithorax. Proc Natl Acad Sci U S A 104:16946–16951PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Chan SK, Wang XA, Mak SS et al (1994) The DNA binding specificity of Ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein. Cell 78:603–615PubMedCrossRefGoogle Scholar
  14. 14.
    Rippe K (1997) Analysis of protein-DNA binding at equilibrium. B I F Futura 12:20–26Google Scholar
  15. 15.
    Bondos SE (2006) Methods for measuring protein aggregation. Curr Anal Chem 2:157–170CrossRefGoogle Scholar
  16. 16.
    Churion KA, Bondos SE (2012) Identifying solubility-promoting buffers for intrinsically disordered proteins prior to purification. Methods Mol Biol 896:415–427PubMedGoogle Scholar
  17. 17.
    Liu Y, Matthews KS, Bondos SE (2008) Multiple intrinsically disordered sequences alter DNA binding by the homeodomain of the Drosophila Hox protein Ultrabithorax. J Biol Chem 283:20874–20887PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Wong EYM, Wang XA, Mak SS et al (2011) Hoxb3 negatively regulates Hoxb1 expression in mouse hindbrain patterning. Dev Biol 352:382–392PubMedCrossRefGoogle Scholar
  19. 19.
    Shen WF, Rozenfeld S, Kwong A et al (1999) HOXA9 forms triple complexes with PBX2 and MEIS1 in myeloid cells. Mol Cell Biol 19:3051–3061PubMedCentralPubMedGoogle Scholar
  20. 20.
    Galant R, Walsh CM, Carroll SB (2002) Hox repression of a target gene: extradenticle-independent, additive action through multiple monomer binding sites. Development 129:3115–3126PubMedGoogle Scholar
  21. 21.
    Li L, von Kessler D, Beachy PA et al (1996) pH-dependent enhancement of DNA binding by the Ultrabithorax homeodomain. Biochemistry 35:9832–9839PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Kelly Churion
    • 1
  • Ying Liu
    • 2
  • Hao-Ching Hsiao
    • 1
  • Kathleen S. Matthews
    • 3
  • Sarah E. Bondos
    • 4
    • 3
    Email author
  1. 1.Department of Molecular and Cellular MedicineTexas A&M Health Science CenterCollege StationUSA
  2. 2.Medical Genomics Laboratory, UAB School of MedicineUniversity of Alabama at BirminghamBirminghamUSA
  3. 3.Department of Biochemistry and Cell BiologyRice UniversityHoustonUSA
  4. 4.Department of Molecular and Cellular MedicineTexas A&M Health Science CenterCollege StationUSA

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