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首页> 外文期刊>Electrophoresis: The Official Journal of the International Electrophoresis Society >Stabilizing labile DNA-protein complexes in polyacrylamide gels.
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Stabilizing labile DNA-protein complexes in polyacrylamide gels.

机译：稳定聚丙烯酰胺凝胶中不稳定的DNA-蛋白质复合物。

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摘要

The electrophoretic mobility-shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA-protein interactions. EMSA, as standardly practiced today, works well for complexes with association binding constants K(a)>10(9) M(-1) under normal conditions of salt and pH. Many DNA-protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using EMSA. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein-DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and CE is straightforward.
机译：电泳迁移率变动分析（EMSA）是分子生物学中用于测量DNA-蛋白质相互作用的最流行工具之一。如今，EMSA在盐和pH值正常条件下，对于缔合结合常数K（a）> 10（9）M（-1）的配合物，效果很好。许多DNA-蛋白质复合物不够稳定，因此它们在通过凝胶基质时会解离，从而产生难以可靠定量的条带。在这项工作中，我们证明了向聚丙烯酰胺凝胶中添加渗透剂三甘醇可显着稳定具有非特异性DNA序列的不稳定限制性核酸内切酶EcoRI复合物，从而能够使用EMSA定量结合。通过向凝胶基质中添加渗透压而产生的技术上的重大改进极大地扩展了蛋白质-DNA复合物的结合常数范围，可以使用这种广泛使用的测定方法进行研究。将该方法扩展到用于分离结合和游离组分的其他技术（例如凝胶色谱和CE）很简单。

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