Several methods are available to visualize and assess the kinetics and efficiency of elemental steps of protein biosynthesis. However, each of these methods has its own limitations. Here, we present a novel, simple and convenient tool for monitoring stepwise in vitro translation initiated by BODIPY-Met-tRNA. Synthesis and release of very short, 1–7 amino acids, BODIPY-labeled peptides, can be monitored using urea–polyacrylamide gel electrophoresis. Very short BODIPY-labeled oligopeptides might be resolved this way, in contrast to widely used Tris-tricine gel electrophoresis, which is suitable to separate peptides larger than 1 kDa. The method described in this manuscript allows one to monitor the steps of translation initiation, peptide transfer, translocation, and termination as well as their inhibition at an unprecedented single amino acid resolution.

中文翻译：

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通过荧光标记小肽凝胶电泳监测体外翻译基本步骤的简单工具


有几种方法可用于可视化和评估蛋白质生物合成基本步骤的动力学和效率。然而，这些方法都有其自身的局限性。在这里，我们提出了一种新颖、简单且方便的工具，用于监测 BODIPY-Met-tRNA 启动的逐步体外翻译。非常短的 1-7 个氨基酸、BODIPY 标记肽的合成和释放可以使用尿素聚丙烯酰胺凝胶电泳进行监测。与广泛使用的 Tris-tricine 凝胶电泳相比，非常短的 BODIPY 标记寡肽可以通过这种方式进行解析，Tris-tricine 凝胶电泳适合分离大于 1 kDa 的肽。本手稿中描述的方法允许人们以前所未有的单氨基酸分辨率监测翻译起始、肽转移、易位和终止的步骤以及它们的抑制。