Detection and Isolation of DNA-Binding Proteins Using Single-Pulse Ultraviolet Laser Crosslinking

✍ Scribed by D.T. Ho; D.M. Sauve; M. Roberge

• Book ID: 102965083
• Publisher: Elsevier Science
• Year: 1994
• Tongue: English
• Weight: 675 KB
• Volume: 218
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1994.1174

✦ Synopsis

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.