Steiner Sebastian, Pfannschmidt Thomas
Institut für Allgemeine Botanik und Pflanzenphysiologie, Lehrstuhl Pflanzenphysiologie Friedrich-Schiller-Universität Jena, Dornburger Str. 159, 07743, Jena, Germany.
Methods Mol Biol. 2009;479:273-89. doi: 10.1007/978-1-59745-289-2_18.
Changes in gene expression mediated by DNA-binding protein factors are a crucial part of many signal transduction pathways. Generally, these regulatory proteins are low abundant and thus their purification and characterisation is labour- and time-intensive. Here we describe a workflow for purification, characterisation and identification of DNA-binding proteins. We show the use of a fluorescence-based electrophoretic mobility shift assay (fEMSA) and describe its advantages for a rapid and convenient screening for regulatory cis-elements. This involves a crude enrichment of nucleic acid binding proteins by heparin-Sepharose chromatography and the characterisation of fractions using overlapping fluorescence-labelled DNA probes spanning the promoter region of interest. The determined protein-binding sites can then be used for sequence-specific DNA-affinity chromatography to purify specifically interacting proteins. Finally, the DNA-binding complexes can be characterised and identified using two-dimensional EMSA, UV-cross-linking and mass spectrometry.
由DNA结合蛋白因子介导的基因表达变化是许多信号转导途径的关键部分。通常，这些调节蛋白丰度较低，因此其纯化和表征工作既费力又耗时。在此，我们描述了一种用于纯化、表征和鉴定DNA结合蛋白的工作流程。我们展示了基于荧光的电泳迁移率变动分析（fEMSA）的应用，并描述了其在快速便捷筛选调控顺式元件方面的优势。这包括通过肝素-琼脂糖凝胶色谱法对核酸结合蛋白进行粗富集，以及使用跨越感兴趣启动子区域的重叠荧光标记DNA探针来表征各组分。然后，所确定的蛋白结合位点可用于序列特异性DNA亲和色谱法，以纯化特异性相互作用的蛋白。最后，可使用二维EMSA、紫外交联和质谱对DNA结合复合物进行表征和鉴定。
