Gurevich Igor, Zhang Carmen, Aneskievich Brian J
Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT, USA.
Methods Mol Biol. 2010;585:147-58. doi: 10.1007/978-1-60761-380-0_11.
Detection of in vitro protein-DNA interaction is one of many investigational analyses for transcription factor regulation of gene promoters. The electrophoretic mobility shift assay (EMSA) has proven widely popular in this respect by integrating individual techniques (protein isolation, nucleic acid radiolabeling, and gel electrophoresis) into one protocol. However, relatively short DNA oligomers are often used which in many cases presupposes what one sequence out of a promoter of possibly thousands of base pairs is the candidate region interacting with a transcription factor. This can be an experimentally distressing situation when multiple putative binding sites of less than perfect consensus may be present making selection of any one or even a few potential sites uncertain or when one is seeking improved throughput as opposed to a one factor:one oligomer approach for in vitro testing of algorithm-predicted binding sites. We describe here our use and refinement of a variant EMSA that can employ multiple and relatively long (up to 1000 bp) probes of promoter sequence in one binding reaction for interaction with nuclear proteins in general and individual transcription factors in particular. We provide labeling and electrophoresis methods suitable for such probes and also highlight the mobility shift differences one can expect with the variant probe method.
体外蛋白质 - DNA 相互作用的检测是用于研究基因启动子转录因子调控的众多分析方法之一。在这方面，电泳迁移率变动分析（EMSA）通过将个体技术（蛋白质分离、核酸放射性标记和凝胶电泳）整合到一个实验方案中，已被证明广泛流行。然而，通常使用的是相对较短的 DNA 寡聚物，在许多情况下，这就预先假定了在可能长达数千个碱基对的启动子中，哪一个序列是与转录因子相互作用的候选区域。当存在多个共识性不太完美的假定结合位点，使得选择任何一个甚至几个潜在位点都不确定时，或者当人们寻求提高通量，而不是采用一个因子：一个寡聚物的方法来体外测试算法预测的结合位点时，这可能是一种在实验上令人苦恼的情况。我们在此描述了一种改良的 EMSA 的使用方法，该方法可以在一次结合反应中采用多个相对较长（长达 1000 bp）的启动子序列探针，用于与一般的核蛋白尤其是单个转录因子相互作用。我们提供了适用于此类探针的标记和电泳方法，并强调了使用改良探针方法时可能出现的迁移率变动差异。
