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Anal Biochem. 1994 May 1;218(2):248-54.

Detection and isolation of DNA-binding proteins using single-pulse ultraviolet laser crosslinking.

Author information

  • 1Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.

Abstract

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.

PMID:
8074276
[PubMed - indexed for MEDLINE]
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