DETECTION AND ISOLATION OF DNA-BINDING PROTEINS USING SINGLE-PULSE ULTRAVIOLET-LASER CROSS-LINKING

Ultraviolet laser crosslinking of proteins to DNA is a potentially pow erful tool for studying protein-nucleic acid interactions in vitro and in vivo. We describe a simple, rapid, and reliable procedure to detec t 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 purifie d preparations. It is also well suited for kinetic analysis, and can d etect transient protein-DNA interactions as well as interactions that are labile in band-shift gels. We show that the method is generally ap plicable to DNA-binding proteins. In addition, we describe a technique to isolate crosslinked protein-DNA complexes from crude extracts in o ne rapid step, using biotinylated DNA probes. Ultraviolet laser crossl inking is a useful alternative or complement to commonly used techniqu es for the detection and characterization of DNA-binding proteins. (C) 1994 Academic Press, Inc.