Mariana Yaneva and Paul Tempst Summary

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 phos-phocellulose (P11) chromatography, followed by a series of positive/negative selections on wild-type and site-mutated ligand DNA in a magnetic microparticulate format. DNA-affinity 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.

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