Solid phase technology improves coupled gel shift/footprinting analysis.
Solid phase technology improves coupled gel shift/footprinting analysis.
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固相技术改进了耦合凝胶位移/足迹分析。
作者：
E. Ragnhildstveit;A. Fjose;P. Becker;J. Quivy
为了通过偶联凝胶位移/足迹法分析蛋白质-DNA相互作用，需要从聚丙烯酰胺凝胶中提取DNA片段，随后在高分辨率凝胶上分离。由于提取的DNA中的杂质，通常不能实现单核苷酸分辨率。我们现在描述一种改进的实验策略，其采用DNA片段与固体支持物的瞬时偶联，以定量、快速和可靠地提取高纯度的DNA。作为一个例子，我们描述了我们的协议的应用程序的“在凝胶足迹”由铜菲咯啉。该方法也可应用于化学干扰分析。
For the analysis of protein-DNA interactions by coupled gel-shift/footprinting, DNA fragments need to be extracted from polyacrylamide gels and subsequently separated on high resolution gels. Due to impurities in the extracted DNA, single nucleotide resolution is frequently not achieved. We now describe an improved experimental strategy that employs transient coupling of DNA fragments to a solid support in order to extract DNA of high purity quantitatively, rapidly and reliably. As an example, we describe the application of our protocol to the 'in-gel footprinting' by copper phenanthroline. The method should also find application to the chemical interference assays.