Advertisement

Identification of Nucleic Acid Binding Proteins Using Nondenaturing Sodium Decyl Sulfate Polyacrylamide Gel Electrophoresis (SDecS-PAGE)

  • Robert E. Akins
  • Rocky S. Tuan

Abstract

Methods for the identification and characterization of nucleic acid binding proteins, such as DNA binding transcription factors, typically involve gel retardation assays (1,2) or Southwestern analysis (3). Gel retardation assays allow the detection of DNAbinding factors by assessing the degree to which protein binding affects the electrophoretic mobility of specific DNA sequences. One drawback of gel retardation assays is that only the presence of the protein is indicated; specific information concerning protein molecular weight, or other characteristics, is obtained only through additional methods. Southwestern analysis detects DNA binding proteins through the use of nucleic acid probes applied to protein blots prepared from sodium dodecyl sulfate (SDS) gels. The Southwestern technique relies on the limited ability of proteins to renature after SDS-gel electrophoresis and does not specifically identify binding by protein complexes, which are dissociated during sample processing.

Keywords

Sodium Dodecyl Sulfate Mixed Micelle Coomassie Brilliant Blue Nucleic Acid Binding Nucleic Acid Probe 
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.
    Fried, M. and Crothers, D. M. (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids. Res. 9, 6505–6525.PubMedCrossRefGoogle Scholar
  2. 2.
    Garner, Revzin, A. (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 9, 3047–3060.PubMedCrossRefGoogle Scholar
  3. 3.
    Bowen, B., Steinberg, J., Laemmli, U. K., and Weintraub, H. (1980) The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 8, 1–20PubMedCrossRefGoogle Scholar
  4. 4.
    Ornstein, L. (1964) Disc electrophoresis I: Background and theory. Ann. NY Acad. Sci. 121, 321–349PubMedCrossRefGoogle Scholar
  5. 5.
    Davis, B. J. (1964) Disc electrophoresis II: Method of application to human serum proteins. Ann. NYAcad. Sci. 121, 404–27CrossRefGoogle Scholar
  6. 6.
    Akins, R. E. and Tuan, R. S. (1994) Separation of proteins using cetyltrimethylammonium bromide discontinuous gel electrophoresis. Mol. Biotechnol. 1, 211–228.PubMedCrossRefGoogle Scholar
  7. 7.
    Akins, R. E., Levin, P. M., and Tuan, R. S. (1992) Cetyltrimethylammonium bromide discontinuous gel electrophoresis: M r-based separation of proteins with retention of enzymatic activity. Analyt. Biochem. 202, 172–178.PubMedCrossRefGoogle Scholar
  8. 8.
    Akin, D., Shapira, R., and Kinkade, J. M. (1985) The determination of molecular weights of biologically active proteins by cetyltrimethyl ammonium bromide-polyacrylamide gel electrophoresis. Analyt. Biochem. 145, 170–176.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Robert E. Akins
    • 1
  • Rocky S. Tuan
    • 1
  1. 1.Nemours Biomedical Research Program, A.I. duPont Hospital for ChildrenWilmington

Personalised recommendations