Detection and isolation of DNA-binding proteins using single-pulse ultraviolet laser crosslinking.
Ultraviolet laser crosslinking of proteins to DNA is a potentially powerful tool for studying protein-nucleic acid interactions in vitro and in vivo. We describe a simple, rapid, and reliable procedure to detect protein-DNA complexes using crosslinking with a single 5-ns pulse of 266-nm light from a uv laser. The method provides an estimate of the molecular mass of DNA-binding proteins in crude extracts or in purified preparations. It is also well suited for kinetic analysis, and can detect transient protein-DNA interactions as well as interactions that are labile in band-shift gels. We show that the method is generally applicable to DNA-binding proteins. In addition, we describe a technique to isolate crosslinked protein-DNA complexes from crude extracts in one rapid step, using biotinylated DNA probes. Ultraviolet laser crosslinking is a useful alternative or complement to commonly used techniques for the detection and characterization of DNA-binding proteins.
Ho
D T
DT
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Sauvé
D M
DM
Roberge
M
M
D001483:Base Sequence; D002467:Cell Nucleus; D003432:Cross-Linking Reagents; D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000528:radiation effects; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000528:radiation effects; D007834:Lasers; D008969:Molecular Sequence Data; D012097:Repressor Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000528:radiation effects; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D034501:Telomere-Binding Proteins; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D034501:Telomere-Binding Proteins; C074461:RIF1 protein, S cerevisiae; D004247:DNA