Identification of FANCA interacting proteins in mammalian cells using tandem affinity purification and mass spectrometry

Sarah L. Conner, Mu Wang

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Tandem affinity purification (TAP) allows for the isolation of protein complexes under close-to-physiological conditions for subsequent protein identification by mass spectrometry. Although TAP has been successfully applied to yeast system, there are only a few reports in mammalian cells. In this study, the gene fanca was cloned into the commercially available TAP construct from Stratagene and transiently transfected into human embryonic kidney cells (Hek 293). The FANCA interacting proteins were TAP purified and subsequently identified by tandem mass spectrometry under both mitomycin C (MMC) treated and non-treated conditions. Several novel protein-protein interactions were identified by liquid chromatography (LC) tandem mass spectrometry (MS) under both conditions. The interaction between FANCA and Huntingtin (HTT), which was induced by MMC treatment, was also confirmed by western blot analysis. Although more studies need to be done to determine the biological implications of these interactions, this study provides a useful method for understanding protein functions through identification of protein-protein interactions.

Original languageEnglish (US)
Pages (from-to)143-153
Number of pages11
JournalScientific Research and Essays
Volume3
Issue number4
StatePublished - Dec 1 2008

Keywords

  • Fanconi anemia
  • Mass spectrometry
  • Proteomics
  • Tandem affinity purification

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)
  • Engineering(all)
  • Physics and Astronomy(all)

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