Gene Mapping, Discovery, and Expression

Volume 338 of the series Methods in Molecular Biology pp 291-303

Isolation and Mass Spectrometry of Specific DNProteins

  • Mariana YanevaAffiliated withMemorial Sloan-Kettering Cancer Center
  • , Paul TempstAffiliated withMolecular Biology Program, Memorial Sloan-Kettering Cancer Center

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A subset of the proteome that binds to specific DNA sequences is at the center of genome function, integrity, and dynamics. We present a detailed protocol that allows the isolation of any specific DNA binding protein and its subsequent identification by mass spectrometry. The procedure involves prefractionation of crude nuclear extract by phosphocellulose (P11) chromatography, followed by a series of positive/negative selections on wild-type and site-mutated ligand DNA in a magnetic microparticulate format. DNAaffinity capture requires concatamerized and biotinylated ligand, selective salt conditions, and improved competitor DNAs. The amount of protein(s) captured on DNA-magnetic beads is generally sufficient for successful MALDI-TOF and TOF/TOF MS-based protein identification. As an example, we describe the procedures used to isolate and identify four specific transcription factors from 2 ↔ 109 promyelocytic NB4 cells.

Key Words

DNA binding affinity capture magnetic beads transcription factors mass spectrometry