Protection analysis (or "footprinting") of specific protein-DNA complexes in crude nuclear extracts using methidiumpropyl-EDTA-iron (II).

N. F. Landolfi, X. M. Yin, J. D. Capra, P. W. Tucker

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Endonuclease protection or "footprinting" analysis is a powerful technique for identifying the nucleotides involved in a protein-DNA interaction. DNase I is the most often employed endonucleolytic agent; however, this endonuclease does not exhibit the true nonsequence-specific cleavage desired for this type of analysis. Methidiumpropyl-EDTA (MPE) is a synthetic DNA intercalator that cleaves DNA in the presence of ferrous ion and oxygen. Cleavage by MPE exhibits no sequence specificity, a characteristic that makes this reagent better suited for protection analysis. Here we report a generally applicable technique for MPE protection (or "footprinting") analysis of specific DNA-protein complexes from a crude nuclear extract. We have used this method to identify the nucleotides of the immunoglobulin (Ig) heavy chain promoter region that are involved in complex formation with a protein that binds the octameric sequence ATGCAAAT, and we compare our results to those obtained previously using DNase I.

Original languageEnglish (US)
Pages (from-to)500-504
Number of pages5
JournalBioTechniques
Volume7
Issue number5
StatePublished - May 1 1989

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

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