Running-buffer composition influences DNA-protein and protein-protein complexes detected by electrophoretic mobility-shift assay (EMSA)
Running-buffer composition influences DNA-protein and protein-protein complexes detected by electrophoretic mobility-shift assay (EMSA)
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DOI：
10.1042/ba20010007
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发表时间：
2001-06-01
影响因子：
2.8
通讯作者：
Schweizer, M
中科院分区：
文献类型：
--
作者：
Roder, K;Schweizer, M
凝胶迁移试验是一种快速、极其灵敏、技术简单且广泛使用的用于研究核酸-蛋白质相互作用的方法，其基于蛋白质与DNA或RNA片段的结合通常导致片段在非变性凝胶中的电泳迁移率降低的观察结果。在这里，我们报告了运行缓冲液的关键作用，并表明其重要性与影响复合物形成和稳定性的其他因素，如结合缓冲液，温度，非特异性竞争剂或凝胶浓度和/或组合物同等重要。我们证明了含有广泛表达的转录因子Sp I结合位点的寡核苷酸的结合模式的差异（刺激蛋白I）、NF-Y（核因子Y）和USF（上游刺激蛋白），其取决于所用运行缓冲液的离子强度。此外，我们使用在大肠杆菌中表达的重组谷胱甘肽S-转移酶-Spl融合蛋白显示了甘氨酸浓度对Sp I结合的影响。
The gel-shift assay is a rapid, extremely sensitive, technically simple and widely used method for investigating nucleic acid-protein interaction based on the observation that binding of protein to DNA or RNA fragments usually leads to a reduction in the electrophoretic mobility of the fragment in non-denaturing gels. Here we report on the critical role of the running buffer and show that its importance ranks equally with other factors affecting complex formation and stability such as binding buffer, temperature, non-specific competitor or gel concentration and/or composition, We demonstrate differences in the binding patterns obtained with oligonucleotides containing binding sites for the ubiquitously expressed transcription factors Sp I (stimulatory protein I), NF-Y (nuclear factor Y) and USF (upstream stimulatory protein), which are dependent on the ionic strength of the running buffer used. Furthermore, we show the influence of:glycine concentration on Sp I binding using recombinant glutathione S-transferase-Spl fusion protein expressed in Escherichia coli.