Isolation and mass spectrometry of specific DNA binding proteins.
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. 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 x 10(9) promyelocytic NB4 cells.
Yaneva
Mariana
M
Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Tempst
Paul
P
D015153:Blotting, Western; D002460:Cell Line; D002482:Cellulose / Q000031:analogs & derivatives; D002845:Chromatography; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D008280:Magnetics; D013058:Mass Spectrometry / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000737:chemistry / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D008024:Ligands; D014157:Transcription Factors; D002482:Cellulose; D004247:DNA; C008881:phosphocellulose