Elucidating protein: DNA complex by oligonucleotide DNA affinity purification

Teddy T.C. Yang, Chi Wing Chow

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations

Abstract

Transcription factors recruit a wide variety of associated co-factors to regulate gene expression. These co-factors include protein kinases, phosphatases, deacetylases, methylases, and ubiquitin ligases, etc. To identify novel protein kinases associated with transcription factor NFAT, we took advantage of the increased ability of DNA binding and used an oligonucleotide affinity-binding approach. Coupling with in-gel kinase assays to detect phosphotransferase activity, we were able to identify p90 ribosomal S6 kinase (RSK) and p70 S6 kinase (S6K) that are present in the NFAT:DNA complex. We further demonstrated that RSK and S6K binds to and physically interacts with NFATc4. Similar oligonucleotide affinity-binding approach can be coupled with other enzymatic reactions, such as dephosphorylation, deacetylation, methylation, ubiquitination, etc. Mass spectrometry can also be carried out to systemically identify these transcription co-factors in the protein:DNA complex. Lastly, gene-specific enhancer elements can also be devised based on their respective sequence to identify distinctive protein:DNA complexes.

Original languageEnglish (US)
Title of host publicationTranscriptional Regulation
Subtitle of host publicationMethods and Protocols
EditorsAles Vancura
Pages75-84
Number of pages10
DOIs
StatePublished - Jan 2 2012

Publication series

NameMethods in Molecular Biology
Volume809
ISSN (Print)1064-3745

Keywords

  • Oligonucleotide DNA affinity purification
  • Protein kinases
  • Transcription activation complex
  • Transcription co-factors
  • Transcription factor NFAT

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Yang, T. T. C., & Chow, C. W. (2012). Elucidating protein: DNA complex by oligonucleotide DNA affinity purification. In A. Vancura (Ed.), Transcriptional Regulation: Methods and Protocols (pp. 75-84). (Methods in Molecular Biology; Vol. 809). https://doi.org/10.1007/978-1-61779-376-9_5