Skip to main content
SpringerLink
Log in

Purification of protein–DNA complexes by native gel electrophoresis for electron microscopy study

  • Methods
  • Published:
Moscow University Biological Sciences Bulletin Aims and scope Submit manuscript

Abstract

Electrophoretic separation under native conditions may be used for purification of protein molecules and their complexes with DNA and other ligands. Here, we employed this approach to separate protein-DNA complexes with a molecular weight of approximately 200 kDa: mono- and dinucleosomes. The purified mononucleosomes were subjected to single particle electron microscopy study using negative stain contrasting, and the two-dimensional projections of the nucleosomes at 25 Å resolution were obtained. A comparison of the nucleosome projections before and after separation in the native PAGE revealed different orientation of particles on the carbon film.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Clapier, C. and Cairns, B., The biology of chromatin remodeling complexes, Annu. Rev. Biochem., 2009, vol. 78, pp. 273–304.

    Article  CAS  PubMed  Google Scholar 

  2. Vignali, M., Hassan, A., Neely, K., and Workman, J., ATP-dependent chromatin-remodeling complexes, Mol. Cell. Biol., 2000, vol. 20, no. 6, pp. 1899–1910.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kornberg, R.D. and Thomas, J.O., Chromatin structure: Oligomers of the histones, Science, 1974, vol. 184, no. 4139, pp. 865–868.

    Article  CAS  PubMed  Google Scholar 

  4. Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F., and Richmond, T.J., Crystal structure of the nucleosome core particle at 2.8 Å resolution, Nature, 1997, vol. 389, no. 6648, pp. 251–260.

    Article  CAS  PubMed  Google Scholar 

  5. Arents, G. and Moudrianakis, E.N., The histone fold: A ubiquitous architectural motif utilized in DNA compaction and protein dimerization, Proc. Natl. Acad. Sci. U.S.A., 1995, vol. 92, no. 24, pp. 11170–11174.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dechassa, M.L., Zhang, B., Horowitz-Scherer, R., Persinger, J., Woodcock, C.L., Peterson, C.L., and Bartholomew, B., Architecture of the SWI/SNFnucleosome complex, Mol. Cell. Biol., 2008, vol. 28, no. 19, pp. 6010–6021.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Feser, J., Truong, D., Das, C., Carson, J.J., Kieft, J., Harkness, T., and Tyler, J.K., Elevated histone expression promotes life span extension, Mol. Cell, 2010, vol. 39, no. 5, pp. 724–735.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ljungman, M. and Hanawalt, P., Efficient protection against oxidative DNA damage in chromatin, Mol. Carcinog., 1992, vol. 5, no. 4, pp. 264–269.

    Article  CAS  PubMed  Google Scholar 

  9. Enright, H.U., Miller, W.J., and Hebbel, R.P., Nucleosomal histone protein protects DNA from iron mediated damage, Nucleic Acids Res., 1992, vol. 20, no. 13, pp. 3341–3346.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. van Heel, M., Gowen, B., Matadeen, R., Orlova, E.V., Finn, R., Pape, T., Cohen, D., Stark, H., Schmidt, R., Schatz, M., and Patwardhan, A., Single-particle electron cryo-microscopy: Towards atomic resolution, Q. Rev. Biophys., 2000, vol. 33, no. 4, pp. 307–369.

    Article  PubMed  Google Scholar 

  11. Knispel, R.W., Kofler, C., Boicu, M., and Nickel, W.B.S., Blotting protein complexes from native gels to electron microscopy grids, Nat. Methods, 2012, vol. 9, no. 2, pp. 182–184.

    Article  CAS  PubMed  Google Scholar 

  12. Pestov, N.A., Gerasimova, N.S., Kulaeva, O.I., and Studitsky, V.M., Structure of transcribed chromatin is a sensor of DNA damage, Sci. Adv., 2015, vol. 1, no. 6.

    Google Scholar 

  13. Gaykalova, D.A., Kulaeva, O.I., Bondarenko, V.A., and Studitsky, V.M., Preparation and analysis of uniquely positioned mononucleosomes, Methods Mol. Biol., 2009, vol. 523, pp. 109–123.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R., and Schatz, M., A new generation of the imagic image processing system, J. Struct. Biol., 1996, vol. 116, no. 1, pp. 17–24.

    Article  PubMed  Google Scholar 

  15. Frank, J., Wagenknecht, T., McEwen, B.F., Marko, M., Hsieh, C.E., and Mannella, C.A., Three-dimensional imaging of biological complexity, J. Struct. Biol., 2002, vol. 138, nos. 1–2, pp. 85–91.

    Article  PubMed  Google Scholar 

  16. Henderson, R., The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules, Q. Rev. Biophys., 1995, vol. 28, no. 2, pp. 171–193.

    Article  CAS  PubMed  Google Scholar 

  17. Glaeser, R.M., How good can cryo-EM become?, Nat. Methods, 2012, vol. 13, no. 1, pp. 28–32.

    Article  Google Scholar 

  18. Asturias, F.J., Chung, W.H., Kornberg, R.D., and Lorch, Y., Structural analysis of the RSC chromatin remodeling complex, Proc. Natl. Acad. Sci. U.S.A., 2002, vol. 99, no. 21, pp. 13477–13480.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Davey, C.A., Sargent, D.F., Luger, K., Maeder, A.W., and Richmond, T.J., Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution, J. Mol. Biol., 2002, vol. 319, no. 5, pp. 1097–1113.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, Moscow State University, Moscow, 119234, Russia

    M. E. Valieva & O. S. Sokolova

  2. Department of Biochemistry, Evdokimov University of Medicine and Dentistry, Moscow, 127473, Russia

    N. I. Derkacheva

Authors
  1. M. E. Valieva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. N. I. Derkacheva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. O. S. Sokolova
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to O. S. Sokolova.

Additional information

Original Russian Text © M.E. Valieva, N.I. Derkacheva, O.S. Sokolova, 2017, published in Vestnik Moskovskogo Universiteta, Seriya 16: Biologiya, 2017, No. 1, pp. 3–8.

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Valieva, M.E., Derkacheva, N.I. & Sokolova, O.S. Purification of protein–DNA complexes by native gel electrophoresis for electron microscopy study. Moscow Univ. Biol.Sci. Bull. 72, 1–5 (2017). https://doi.org/10.3103/S0096392517010059

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.3103/S0096392517010059

Keywords

Search

Navigation