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Protein Blotting of Basic Proteins Resolved on Acid-Urea-Triton-Polyacrylamide Gels

  • Geneviève P. Delcuve
  • James R. Davie

Abstract

The electrophoretic resolution of histones on acetic acid-urea-Triton (AUT) polyacrylamide gels is the method of choice to separate basic proteins, such as histone variants, modified histone species, and high-mobility group proteins 14 and 17 (1-6 and see  Chapters 16 and  17). Basic proteins are resolved in this system on the basis of their size, charge, and hydrophobicity. In previous studies, we analyzed the abundance of ubiquitinated histones by resolving the histones on two-dimensional (AUT into SDS) polyacrylamide gels, followed by their transfer to nitrocellulose membranes, and immunochemical staining of nitrocellulose membranes with an antiubiquitin antibody (7-9). However, transfer of the basic proteins directly from the AUT polyacrylamide gel circumvents the need to run the second-dimension SDS gel and accomplishes the analysis of several histone samples. We have described a method that efficiently transfers basic proteins from AUT polyacrylamide gels to nitrocellulose membranes (10). This method has been used in the immunochemical detection of modified histone, isoforms, and histone H1 subtypes (6,11-13).

Keywords

Nitrocellulose Membrane Basic Protein Equilibration Buffer Histone Variant Transfer Buffer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Urban, M. K., Franklin, S. G., and Zweidler, A. (1979) Isolation and characterization of the histone variants in chicken erythrocytes. Biochemistry 18, 3952–3959.PubMedCrossRefGoogle Scholar
  2. 2.
    Strickland, M., Strickland, W. N., and Von Holt, C. (1981) The occurrence of sperm isohistones H2B in single sea urchins. FEBS Lett. 135, 86–88.PubMedCrossRefGoogle Scholar
  3. 3.
    Meistrich, M. L., Bucci, L. R., Trostle-Weige, P. K., and Brock, W. A. (1985) Histone variants in rat spermatogonia and primary spermatocytes. Dev. Biol. 112, 230–240.PubMedCrossRefGoogle Scholar
  4. 4.
    Waterborg, J. H. (1990) Sequence analysis of acetylation and methylation in two histone H3 variants of alfalfa. J. Biol. Chem. 265, 17, 157–17, 161.Google Scholar
  5. 5.
    Davie, J. R. and Delcuve, G. P. (1991) Characterization and chromatin distribution of the H1 histones and high-mobility-group non-histone chromosomal proteins of trout liver and hepatocellular carcinoma. Bio chem. J. 280, 491–497.Google Scholar
  6. 6.
    Li, W., Nagaraja, S., Delcuve, G. P., Hendzel, M. J., and Davie, J. R. (1993) Effects of histone acetylation, ubiquitination and variants on nucleosome stability. Biochem. J. 296, 737–744.PubMedGoogle Scholar
  7. 7.
    Nickel, B. E., Allis, C. D., and Davie, J. R. (1989) Ubiquitinated histone H2B is preferentially located in transcriptionally active chromatin. Biochemistry 28, 958–963.PubMedCrossRefGoogle Scholar
  8. 8.
    Davie, J. R. and Murphy, L. C. (1990) Level of ubiquitinated histone H2B in chromatin is coupled to ongoing transcription. Biochemistry 29, 4752–4757.PubMedCrossRefGoogle Scholar
  9. 9.
    Davie, J. R., Lin, R., and Allis, C. D. (1991) Timing of the appearance of ubiquitinated histones in developing new macronuclei of Tetrahymena thermophila. Biochem. Cell Biol. 69, 66–71.PubMedCrossRefGoogle Scholar
  10. 10.
    Delcuve, G. P. and Davie, J. R. (1992) Western blotting and immunochemical detection of histones electrophoretically resolved on acid-urera-triton-and sodium dodecyl sulfatepolyacrylamide gels. Analyt. Biochem. 200, 339–341.PubMedCrossRefGoogle Scholar
  11. 11.
    Lee, D. Y., Hayes, J. J., Pruss, D., and Wolffe, A. P. (1993) A positive role for histone acetylation in transcription factor access to nucleosomal DNA. Cell 72, 73–84.PubMedCrossRefGoogle Scholar
  12. 12.
    Davie, J. R. and Murphy, L. C. (1994) Inhibition of transcription selectively reduces the level of ubiquitinated histone H2B in chromatin. Biochem. Biophys. Res. Commun. 203, 344–350.PubMedCrossRefGoogle Scholar
  13. 13.
    Nagaraja, S., Delcuve, G. P., and Davie, J. R. (1995) Differential compaction of transcriptionally competent and repressed chromatin reconstituted with histone H1 subtypes. Biochim. Biophys. Acta 1260, 207–214.PubMedGoogle Scholar
  14. 14.
    Szewczyk, B. and Kozloff, L. M. (1985) A method for the efficient blotting of strongly basic proteins from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose. Analyt. Biochem. 150, 403–407.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Geneviève P. Delcuve
    • 1
  • James R. Davie
    • 1
  1. 1.Manitoba Institute of Cell BiologyWinnipegCanada

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