Ho D T, Sauvé D M, Roberge M
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Anal Biochem. 1994 May 1;218(2):248-54. doi: 10.1006/abio.1994.1174.
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.
蛋白质与DNA的紫外激光交联是一种在体外和体内研究蛋白质 - 核酸相互作用的潜在强大工具。我们描述了一种简单、快速且可靠的程序，使用来自紫外激光的单个5纳秒266纳米光脉冲进行交联来检测蛋白质 - DNA复合物。该方法可估算粗提物或纯化制剂中DNA结合蛋白的分子量。它也非常适合动力学分析，并且能够检测瞬时蛋白质 - DNA相互作用以及在凝胶迁移实验中不稳定的相互作用。我们表明该方法通常适用于DNA结合蛋白。此外，我们描述了一种使用生物素化DNA探针从粗提物中快速一步分离交联的蛋白质 - DNA复合物的技术。紫外激光交联是用于检测和表征DNA结合蛋白的常用技术的一种有用替代方法或补充方法。
