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Identification of Sequence-Specific DNA-Binding Proteins by Southwestern Blotting

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DNA-Protein Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 148))

Abstract

Southwestern blotting was first described by Bowen et al. (1) and was used to identify DNA-binding proteins that specifically interact with a chosen DNA fragment in a sequence-specific manner. In this technique, mixtures of proteins such as crude nuclear extracts or partially purified preparations are first fractionated on a sodium dodecyl sulfate (SDS) denaturing gel; the gel is then equilibrated in a SDS-free buffer to remove detergent and the proteins transferred by electroblotting to an immobilizing membrane. During the transfer the proteins renature and hence DNA-binding proteins may subsequently be detected on the membrane by their ability to bind radiolabeled DNA. Fractionation of crude nuclear extracts on an SDS gel followed by electroblotting and analysis for sequence-specific DNA binding directly on the blot combines the advantages of a high-resolution fractionation step with the ability to rapidly analyze for a large number of different DNA-binding specificities.

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References

  1. Bowen, B., Steinberg, J., Laemmli, U. K., and Weintraub, H. (1980) The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 8, 1–20.

    Article  PubMed  CAS  Google Scholar 

  2. Séguin, C. and Prévost, J. (1988) Detection of a nuclear protein that interacts with a metal regulatory element of the mouse metallothionein 1 gene. Nucleic Acids Res. 16, 10,547–10,560.

    Article  PubMed  Google Scholar 

  3. Jack, R. S., Brown, M. T., and Gehring, W. J. (1983) Protein blotting as a means to detect sequence-specific DNA-binding proteins. Cold Spring Harb. Symp. Quant. Biol. 47(Pt 1), 483–491.

    PubMed  Google Scholar 

  4. Miskimins, W. K., Roberts, M. P., McClelland, A., and Ruddle, F. H. (1985) Use of a protein-blotting procedure and a specific DNA probe to identify nuclear proteins that recognize the promoter region of the transferrin receptor gene. Proc. Natl. Acad. Sci. USA 82, 6741–6744.

    Article  PubMed  CAS  Google Scholar 

  5. Silva, C. M., Tully, D. B., Petch, L. A., Jewell, C. M., and Cidlowski, J. A. (1987) Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA. Proc. Natl. Acad. Sci. USA 84, 1744–1748.

    Article  PubMed  CAS  Google Scholar 

  6. Hughes, E. N., Engelsberg, B. N., and Billings, P. C. (1992) Purification of nuclear proteins that bind to cisplatin-damaged DNA. Identity with high mobility group proteins 1 and 2. J. Biol. Chem. 267, 13,520–13,527.

    PubMed  CAS  Google Scholar 

  7. Wegenka, U. M., Buschmann, J., Lutticken, C., Heinrich, P. C., and Horn, F. (1993) Acute-phase response factor, a nuclear factor binding to acute-phase response elements, is rapidly activated by interleukin-6 at the posttranslational level. Mol. Cell. Biol. 13, 276–288.

    PubMed  CAS  Google Scholar 

  8. Kwast-Welfeld, J., Debelle, I., Walker, P. R., Whitfield, J. F., and Sikorska, M. (1993) Identification of a new cAMP response element-binding factor by southwestern blotting. J. Biol. Chem. 268, 19,581–19,585.

    PubMed  CAS  Google Scholar 

  9. Dhawan, P., Chang, R., and Mehta, K. D. (1997) Identification of essential nucleotides of the FP1 element responsible for enhancement of low density lipoprotein receptor gene transcription. Nucleic Acids Res. 25, 4132–4138.

    Article  PubMed  CAS  Google Scholar 

  10. Villafuerte, B. C., Zhao, W., Herington, A. C., Saffery, R., and Phillips, L. S. (1997) Identification of an insulin-responsive element in the rat insulin-like growth factor-binding protein-3 gene. J. Biol. Chem. 272, 5024–5030.

    Article  PubMed  CAS  Google Scholar 

  11. Wu, J., Jiang, Q., Chen, X., Wu, X. H., and Chan, J. S. (1998) Identification of a novel mouse hepatic 52 kDa protein that interacts with the cAMP response element of the rat angiotensinogen gene. Biochem. J. 329, 623–629.

    PubMed  CAS  Google Scholar 

  12. Melkonyan, H., Hofmann, H. A., Nacken, W., Sorg, C., and Klempt, M. (1998) The gene encoding the myeloid-related protein 14 (MRP14), a calcium-binding protein expressed in granulocytes and monocytes, contains a potent enhancer element in the first intron. J. Biol. Chem. 273, 27,026–27,032.

    Article  PubMed  CAS  Google Scholar 

  13. Carrion, A. M., Mellstrom, B., and Naranjo, J. R. (1998) Protein kinase A-dependent derepression of the human prodynorphin gene via differential binding to an intragenic silencer element. Mol. Cell. Biol. 18, 6921–6929.

    PubMed  CAS  Google Scholar 

  14. Chen, A. and Davis, B. H. (1999) UV irradiation activates JNK and increases alphaI(I) collagen gene expression in rat hepatic stellate cells. J. Biol. Chem. 274, 158–164.

    Article  PubMed  CAS  Google Scholar 

  15. Katsu, Y., Yamashita, M., and Nagahama, Y. (1997) Isolation and characterization of goldfish Y box protein, a germ-cell-specific RNA-binding protein. Eur. J. Biochem. 249, 854–861.

    Article  PubMed  CAS  Google Scholar 

  16. Johnston, K. A., Polymenis, M., Wang, S., Branda, J., and Schmidt, E. V. (1998) Novel regulatory factors interacting with the promoter of the gene encoding the mRNA cap binding protein (eIF4E) and their function in growth regulation. Mol. Cell. Biol. 18, 5621–5633.

    PubMed  CAS  Google Scholar 

  17. Hamann, S. and Stratling, W. H. (1998) Specific binding of Drosophila nuclear protein PEP (protein on ecdysone puffs) to hsp70 DNA and RNA. Nucleic Acids Res. 26, 4108–4115.

    Article  PubMed  CAS  Google Scholar 

  18. Keller, A. D. and Maniatis, T. (1991) Selection of sequences recognized by a DNA binding protein using a preparative Southwestern blot. Nucleic Acids Res. 19, 4675–4680.

    Article  PubMed  CAS  Google Scholar 

  19. Singh, H., Clerc, R. G., and LeBowitz, J. H. (1989) Molecular cloning of sequence-specific DNA binding proteins using recognition site probes. BioTechniques 7, 252–261.

    PubMed  CAS  Google Scholar 

  20. Stuempfle, K. J. and Floros, J. (1997) Caution is advised when cDNA expression libraries are screened by southwestern methodologies. BioTechniques 22, 260–264.

    PubMed  CAS  Google Scholar 

  21. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual (Ford, N., Nolan, C., and Ferguson, M., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  22. Dignam, J. D. (1990) Preparation of extracts from higher eukaryotes. Methods Enzymol. 182, 194–203.

    Article  PubMed  CAS  Google Scholar 

  23. Handen, J. S. and Rosenberg, H. F. (1997) An improved method for southwestern blotting. Front. Biosci. 2, c9–c11.

    CAS  Google Scholar 

  24. Dooley, S., Welter, C., and Blin, N. (1992) Nonradioactive Southwestern analysis using chemiluminescent detection. BioTechniques 13, 540–543.

    PubMed  CAS  Google Scholar 

  25. Schaufele, F., Cassill, J. A., West, B. L., and Reudelhuber, T. (1990) Resolution by diagonal gel mobility shift assays of multisubunit complexes binding to a functionally important element of the rat growth hormone gene promoter. J. Biol. Chem. 265, 14,592–14,598.

    PubMed  CAS  Google Scholar 

  26. Papavassiliou, A. G. and Bohmann, D. (1992) Optimization of the signal-to-noise ratio in south-western assays by using lipid-free BSA as blocking reagent. Nucleic Acids Res. 20, 4365–4366.

    Article  PubMed  CAS  Google Scholar 

  27. Mazen, A., Ménissier-de Murcia, J., Molinete, M., Simonin, F., Gradwohl, G., Poirier, G., et al. (1989) Poly(ADP-ribose) polymerase: a novel finger protein. Nucleic Acids Res. 12, 4689–4698.

    Article  Google Scholar 

  28. Mimori, T., Hardin, J. A., and Steitz, J. A. (1986) Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders. J. Biol. Chem. 261, 2274–2278.

    PubMed  CAS  Google Scholar 

  29. Wold, M. S. (1997) Replication protein A: A heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66, 61–92.

    Article  PubMed  CAS  Google Scholar 

  30. Kannan, P., Yu, Y., Wankhade, S., and Tainsky, M. A. (1999) PolyADP-ribose polymerase is a coactivator for AP-2-mediated transcriptional activation. Nucleic Acids Res. 27, 866–874.

    Article  PubMed  CAS  Google Scholar 

  31. Dear, T. N., Hainzl, T., Follo, M., Nehls, M., Wilmore, H., Matena, K., and Boehm, T. (1997) Identification of interaction partners for the basic-helix-loop-helix protein E47. Oncogene 14, 891–898.

    Article  PubMed  CAS  Google Scholar 

  32. Butler, A. J. and Ordahl, C. P. (1999) Poly(ADP-ribose) polymerase binds with transcription enhancer factor 1 to MCAT1 elements to regulate muscle-specific transcription. Mol. Cell. Biol. 19, 296–306.

    PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, MI

    Simon Labbé

  2. Centre de Recherche en Cancérologie, Université Laval, CHUQ/L’Hotel-Dieu de Québec, Québec, Canada

    Gale Stewart, Olivier LaRochelle & Carl Séguin

  3. Unité Santé et Environment, CHUQ, Pavillon CHUL, Québec, Canada

    Guy G. Poirier

Authors
  1. Simon Labbé
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  2. Gale Stewart
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  3. Olivier LaRochelle
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  4. Carl Séguin
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  5. Guy G. Poirier
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Editor information

Editors and Affiliations

  1. Centre de Recherche en Cancérologie de l’Université Laval, Centre Hopital Universitaire de Québec et Départment de biologie médicale, Université Laval, Québec, QC, Canada

    Tom Moss

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© 2001 Humana Press Inc., Totowa, NJ

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Labbé, S., Stewart, G., LaRochelle, O., Séguin, C., Poirier, G.G. (2001). Identification of Sequence-Specific DNA-Binding Proteins by Southwestern Blotting. In: Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology, vol 148. Humana Press. https://doi.org/10.1385/1-59259-208-2:255

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  • DOI: https://doi.org/10.1385/1-59259-208-2:255

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-625-3

  • Online ISBN: 978-1-59259-208-1

  • eBook Packages: Springer Protocols

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