Advertisement

The Southwestern Assay

  • Jacques Philippe
Protocol
  • 111 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

Determination of cellular phenotypes results from the expression of a limited number of genes whose products interact to establish a unique environment. The mechanisms by which individual cells can selectively express only a few of all the genes in a specific cell have been the focus of intense research during the last 10 yr. It has become apparent that developmental, tissue-specific, and hormone-regulated gene expression is, for the most part, controlled at the level of transcriptional initiation. This involves the interaction of specific DNA binding proteins with control elements present in the gene promoters. To better understand the process of gene transcription, characterization of these DNA binding proteins is a mandatory step.

Keywords

Binding Buffer Ammonium Persulfate Tank Buffer Specific Competitor Nonspecific Competitor 
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.
    Lin, S. Y. and Riggs, A. D. (1975) The general affinity of lac repressor for E. Coli DNA: implication for gene regulation in prokaryotes and eukaryotes. Cell 4, 107–111.PubMedCrossRefGoogle Scholar
  2. 2.
    Galas, D. and Schmidt, A. (1978) DNase footprinting, a simple method for the detection of protein-DNA binding specificity. Nucleic Acids Res. 5, 3157–3170.PubMedCrossRefGoogle Scholar
  3. 3.
    Garner, M. M. and Revzin, A. (1981) A gel electrophoresis method for quantifying binding of proteins to specific DNA regions: applications to components of the E. coli lactose operon regulatory system. Nucleic Acids Res. 9, 3047–3059.PubMedCrossRefGoogle Scholar
  4. 4.
    Chodosh, L. A., Carthew, R. W., and Sharp, P. A. (1986) A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor. Mol. Cell. Biol. 6, 4723–4733.PubMedGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    Vinson, C. R., LaMarco, K. L., Johnson, P. F., Landschulz, W. H., and McKnight, S. L. (1988) In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage. Genes Dev. 2, 801–806.PubMedCrossRefGoogle Scholar
  7. 7.
    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.PubMedCrossRefGoogle Scholar
  8. 8.
    Hoeffler, J. P., Lustbader, J. W., and Chen, C. Y. (1991) Identification of multiple nuclear factors that interact with cAMP-response element-binding protein and activating transcription factor-2 by protein-protein interactions. Mol. Endo. 5, 256–266.CrossRefGoogle Scholar
  9. 9.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  10. 10.
    Dreyfuss, G., Adam, S. A., and Choi, Y. D. (1984) Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription. Mol. Cell. Biol. 4, 415–423.PubMedGoogle Scholar
  11. 11.
    Papavassiliou, A. G. and Bohmann, D. (1992) Optimization of the signal-to-noise ratio in Southwestern assays by using lipid-free BSA as blocking reagent. Nucleic Acids Res. 20, 4365–4366.PubMedCrossRefGoogle Scholar
  12. 12.
    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.PubMedCrossRefGoogle Scholar
  13. 13.
    Jack, R. S., Gehring, W. J., and Brack, C. (1981) Protein component from Drosophila larval nuclei showing sequence specificity for a short region near a major heat shock protein gene. Cell 24, 321–331.PubMedCrossRefGoogle Scholar
  14. 14.
    Miskimins, W. K., Roberts, M. P., McClelland, A., and Ruddle, H. (1985) Use of a protein blotting procedure and a specific DNA probe to identify nuclear proteins that recognizes the region of the transferrin receptor gene. Proc. Natl. Acad. Sci. USA 82, 6741–6744.PubMedCrossRefGoogle Scholar
  15. 15.
    Matsuno, K., Suzuki, T., Takiya, S., and Suzuki, Y. (1989) Complex formation with the fibroin gene enhancer through a protein-protein interaction analyzed by a modified DNA-binding assay. J. Biol Chem. 264, 4599–4604.PubMedGoogle Scholar
  16. 16.
    Hübscher, U. (1987) Double replica Southwestern. Nucleic Acids Res. 15, 5486.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Jacques Philippe
    • 1
  1. 1.Diabetes Unit, Department of MedicineCentre Medical UniversitaireGenevaSwitzerland

Personalised recommendations