Skip to main content

Preparation and analysis of nuclear protein extract from Drosophila melanogaster embryos for studying transcription factors

Abstract

Nuclear protein extract is an important substance for studying different molecular systems of the cell, particularly transcription machinery. The paper briefly describes extraction methods and provides a protocol for the preparation of extract from nuclei of Drosophila embryos using salt extraction. The extraction efficiency of transcription factors and chromatin proteins has been checked under different salt concentrations. The paper also provides data about chromatographic properties of some protein factors regulating gene activity.

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

References

  1. Weiss, S.B, Proc. Natl. Acad. Sci. U. S. A., 1960, vol. 46, pp. 1020–1030.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Hurwitz, J., J. Biol. Chem., 2005, vol. 280, pp. 42477–42485.

    CAS  Article  PubMed  Google Scholar 

  3. Weil, P.A., Segall, J., Harris, B., Ng, S.Y., and Roeder, R.G., J. Biol. Chem., 1979, vol. 254, pp. 6163–6173.

    CAS  PubMed  Google Scholar 

  4. Dignam, J.D., Lebovitz, R.M., and Roeder, R.G, Nucleic Acids Res., 1983, vol. 11, pp. 1475–1489.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Matsui, T., Segall, J., Weil, P.A., and Roeder, R.G., J. Biol. Chem., 1980, vol. 255, pp. 11992–11996.

    CAS  PubMed  Google Scholar 

  6. Rio, D., Robbins, A., Myers, R., and Tjian, R, Proc. Natl. Acad. Sci. U. S. A., 1980, vol. 77, pp. 5706–5710.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  7. Corden, J., Wasylyk, B., Buchwalder, A., Sassone-Corsi, P., Kedinger, C., and Chambon, P, Science, 1980, vol. 209, pp. 1406–1414.

    CAS  Article  PubMed  Google Scholar 

  8. Wasylyk, B., Kedinger, C., Corden, J., Brison, O., and Chambon, P, Nature, 1980, vol. 285, pp. 367–373.

    CAS  Article  PubMed  Google Scholar 

  9. Lee, D.C., Roeder, R.G., and Wold, W.S, Proc. Natl. Acad. Sci. U. S. A., 1982, vol. 79, pp. 41–45.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. Grosveld, G.C., Shewmaker, C.K., Jat, P., and Flavell, R.A, Cell, 1981, vol. 25, pp. 215–226.

    CAS  Article  PubMed  Google Scholar 

  11. Weil, P.A., Luse, D.S., Segall, J., and Roeder, R.G, Cell, 1979, vol. 18, pp. 469–484.

    CAS  Article  PubMed  Google Scholar 

  12. Manley, J.L., Fire, A., Cano, A., Sharp, P.A., and Gefter, M.L, Proc. Natl. Acad. Sci. U. S. A., 1980, vol. 77, pp. 3855–3859.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Dignam, J.D., Martin, P.L., Shastry, B.S., and Roeder, R.G, Methods Enzymol., 1983, vol. 101, pp. 582–598.

    CAS  Article  PubMed  Google Scholar 

  14. Krainer, A.R., Maniatis, T., Ruskin, B., and Green, M.R, Cell, 1984, vol. 36, pp. 993–1005.

    CAS  Article  PubMed  Google Scholar 

  15. Heiermann, R. and Pongs, O, Nucleic Acids Res., 1985, vol. 13, pp. 2709–2730.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Abmayr, S.M., Yao, T., Parmely, T., and Workman, J.L., in Current Protocols in Molecular Biology, Ausubel, F.M., et al., Eds., 2006, chapter 12, unit 1211.

  17. Sanders, M.M., J. Cell Biol., 1978, vol. 79, pp. 97–109.

    CAS  Article  PubMed  Google Scholar 

  18. Teves, S.S. and Henikoff, S, Methods Mol. Biol., 2012, vol. 833, pp. 421–432.

    CAS  Article  PubMed  Google Scholar 

  19. Dreger, M., Bengtsson, L., Schoneberg, T., Otto, H., and Hucho, F, Proc. Natl. Acad. Sci. U. S. A., 2001, vol. 98, pp. 11943–11948.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  20. Otto, H., Dreger, M., Bengtsson, L., and Hucho, F, Eur. J. Biochem./FEBS, 2001, vol. 268, pp. 420–428.

    CAS  Article  Google Scholar 

  21. Villar, D., Flicek, P., and Odom, D.T, Nature Rev. Genet., 2014, vol. 15, pp. 221–233.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Kawasaki, H., Hirose, S., and Ueda, H, Cytotechnology, 2005, vol. 49, pp. 67–70.

    Article  PubMed  PubMed Central  Google Scholar 

  23. O’Neill, B., Rubin Lab Manual, 1990.

    Google Scholar 

  24. Kamakaka, R.T. and Kadonaga, J.T, Methods Cell Biol., 1994, vol. 44, pp. 225–235.

    CAS  Article  PubMed  Google Scholar 

  25. Kamakaka, R.T., Tyree, C.M., and Kadonaga, J.T, Proc. Natl. Acad. Sci. U. S. A., 1991, vol. 88, pp. 1024–1028.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Shechter, D., Dormann, H.L., Allis, C.D., and Hake, S.B, Nature Protocols, 2007, vol. 2, pp. 1445–1457.

    CAS  Article  PubMed  Google Scholar 

  27. Worcel, A., Gargiulo, G., Jessee, B., Udvardy, A., Louis, C., and Schedl, P, Nucleic Acids Res., 1983, vol. 11, pp. 421–439.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Yang, Z., J. Biotechnol., 2009, vol. 144, pp. 12–22.

    CAS  Article  PubMed  Google Scholar 

  29. Rougvie, A.E. and Lis, J.T, Cell, 1988, vol. 54, pp. 795–804.

    CAS  Article  PubMed  Google Scholar 

  30. Vorobyeva, N.E., Soshnikova, N.V., Kuzmina, J.L., Kopantseva, M.R., Nikolenko, J.V., Nabirochkina, E.N., Georgieva, S.G., and Shidlovskii, Y.V, Cell Cycle, 2009, vol. 8, pp. 2152–2156.

    CAS  Article  PubMed  Google Scholar 

  31. Vorobyeva, N.E., Soshnikova, N.V., Nikolenko, J.V., Kuzmina, J.L., Nabirochkina, E.N., Georgieva, S.G., and Shidlovskii, Y.V, Proc. Natl. Acad. Sci. U. S. A., 2009, vol. 106, pp. 11049–11054.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Gurskiy, D., Orlova, A., Vorobyeva, N., Nabirochkina, E., Krasnov, A., Shidlovskii, Y., Georgieva, S., and Kopytova, D, Nucleic Acids Res., 2012, vol. 40, pp. 10689–10700.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Kopytova, D., Popova, V., Kurshakova, M., Shidlovskii, Y., Nabirochkina, E., Brechalov, A., Georgiev, G., and Georgieva, S., Nucleic Acids Res., 2016 (in press).

    Google Scholar 

  34. Kopytova, D.V., Orlova, A.V., Krasnov, A.N., Gurskiy, D.Y., Nikolenko, J.V., Nabirochkina, E.N., Shidlovskii, Y.V., and Georgieva, S.G, Genes Dev., 2010, vol. 24, pp. 86–96.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Panov, V.V., Kuzmina, J.L., Doronin, S.A., Kopantseva, M.R., Nabirochkina, E.N., Georgieva, S.G., Vorobyeva, N.E., and Shidlovskii, Y.V, Nucleic Acids Res., 2012, vol. 40, pp. 2445–2453.

    CAS  Article  PubMed  Google Scholar 

  36. Vorobyeva, N.E., Nikolenko, J.V., Krasnov, A.N., Kuzmina, J.L., Panov, V.V., Nabirochkina, E.N., Georgieva, S.G., and Shidlovskii, Y.V, Cell Cycle, 2011, vol. 10, pp. 1821–1827.

    CAS  Article  PubMed  Google Scholar 

  37. Thomas, M.C. and Chiang, C.M, Crit. Rev. Biochem. Mol. Biol., 2006, vol. 41, pp. 105–178.

    CAS  Article  PubMed  Google Scholar 

  38. Maksimenko, O. and Georgiev, P, Front. Genet, 2014, vol. 5, p. 28.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Grunberg, S. and Hahn, S, Trends Biochem. Sci., 2013, vol. 38, pp. 603–611.

    CAS  Article  PubMed  Google Scholar 

  40. Levine, M. and Tjian, R, Nature, 2003, vol. 424, pp. 147–151.

    CAS  Article  PubMed  Google Scholar 

  41. Papai, G., Weil, P.A., and Schultz, P, Curr. Opin. Genet. Dev., 2011, vol. 21, pp. 219–224.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Nagy, Z. and Tora, L, Oncogene, 2007, vol. 26, pp. 5341–5357.

    CAS  Article  PubMed  Google Scholar 

  43. Robinson, P.J., Trnka, M.J., Pellarin, R., Greenberg, C.H., Bushnell, D.A., Davis, R., Burlingame, A.L., Sali, A., and Kornberg, R.D., eLife, 2015, vol.4.

  44. Kal, A.J., Mahmoudi, T., Zak, N.B., and Verrijzer, C.P, Genes Dev., 2000, vol. 14, pp. 1058–1071.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Mohrmann, L., Langenberg, K., Krijgsveld, J., Kal, A.J., Heck, A.J., and Verrijzer, C.P, Mol. Cell. Biol., 2004, vol. 24, pp. 3077–3088.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. Janody, F., Martirosyan, Z., Benlali, A., and Treisman, J.E, Development, 2003, vol. 130, pp. 3691–3701.

    CAS  Article  PubMed  Google Scholar 

  47. Maksimenko, O., Kyrchanova, O., Bonchuk, A., Stakhov, V., Parshikov, A., and Georgiev, P, Epigenetics: Official Journal of the DNA Methylation Society, 2014, vol. 9, pp. 1261–1270.

    Article  Google Scholar 

  48. Vorobyeva, N.E., Mazina, M.U., Golovnin, A.K., Kopytova, D.V., Gurskiy, D.Y., Nabirochkina, E.N., Georgieva, S.G., Georgiev, P.G., and Krasnov, A.N, Nucleic Acids Res., 2013, vol. 41, pp. 5717–5730.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Georgiev, P.G. and Gerasimova, T.I, Genetika, 1989, vol. 25, pp. 1409–1419.

    CAS  PubMed  Google Scholar 

  50. Dosantos, G., Schroeder, A.J., Goodman, J.L., Strelets, V.B., Crosby, M.A., Thurmond, J., Emmert, D.B., and Gelbart, W.M., Nucleic Acids Res., 2015, vol. 43, pp. D690–D697.

    Article  Google Scholar 

  51. Biggin, M.D. and Tjian, R, Cell, 1988, vol. 53, pp. 699–711.

    CAS  Article  PubMed  Google Scholar 

  52. Neri, L.M., Riederer, B.M., Valmori, A., Capitani, S., and Martelli, A.M., J. Histochem. Cytochem.: Official Journal of the Histochemistry Society, 1997, vol. 45, pp. 1317–1328.

    CAS  Article  Google Scholar 

  53. Hilderson, H., Voets, R., Lagrou, A, Van Dessel, G., and Dierick, W, Int. J. Biochem., 1984, vol. 16, pp. 31–37.

    CAS  Article  PubMed  Google Scholar 

  54. Go, Y.M. and Jones, D.P, Biochim. Biophys. Acta, 2008, vol. 1780, pp. 1273–1290.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to A. V. Shaposhnikov.

Additional information

Original Russian Text © A.V. Shaposhnikov, L.A. Lebedeva, E.S. Chernioglo, Z.M. Kachaev, A. Abdrakhmanov, Yu.V. Shidlovskii, 2016, published in Bioorganicheskaya Khimiya, 2016, Vol. 42, No. 6, pp. 712–721.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Shaposhnikov, A.V., Lebedeva, L.A., Chernioglo, E.S. et al. Preparation and analysis of nuclear protein extract from Drosophila melanogaster embryos for studying transcription factors. Russ J Bioorg Chem 42, 646–654 (2016). https://doi.org/10.1134/S1068162016060108

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1134/S1068162016060108

Keywords

  • nuclear protein extract
  • Drosophila melanogaster
  • embryos
  • transcription factor