Graduate School of Science and Engineering, Saitama University, Sakura-ku, Saitama 338-8570, Japan.
Anal Biochem. 2013 Mar 1;434(1):93-5. doi: 10.1016/j.ab.2012.10.041. Epub 2012 Nov 10.
In this paper, we demonstrate a novel pull-down method that dramatically reduces the cost and preparation time of a bait protein by cell-free translation with a puromycin linker. With the C-terminus of the bait protein linked to biotin through a puromycin molecule after the translation reaction and subsequent mRNA degradation by RNase, the prey protein was easily pulled down by streptavidin-coated magnetic beads in a test tube. Three fluorescent prey protein types were tested and confirmed by gel electrophoresis to be pulled down easily and rapidly, depending on their affinity.
在本文中，我们展示了一种新颖的下拉方法，通过无细胞翻译并用嘌呤霉素接头，大大降低了诱饵蛋白的成本和制备时间。在翻译反应后，诱饵蛋白的 C 端通过嘌呤霉素分子与生物素连接，随后通过 RNase 降解 mRNA，使猎物蛋白在试管中很容易通过链霉亲和素包被的磁珠下拉。通过凝胶电泳测试和确认了三种荧光型猎物蛋白，它们根据亲和力很容易且快速地被拉下。
