Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia.
Elife. 2018 Aug 10;7:e36530. doi: 10.7554/eLife.36530.
We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.
我们描述了 Ribo Mega-SEC，这是一种使用体积排阻色谱和 uHPLC 分离和生化分析哺乳动物多核糖体和核糖体亚基的强大方法。使用来自细胞或组织的提取物，多核糖体可以在 15 分钟内从样品注射到馏分收集进行分离。Ribo Mega-SEC 表明，翻译核糖体主要存在于人细胞系和小鼠肝组织中的多核糖体复合物中。在处理之间（例如，细胞对氨基酸饥饿的反应），很容易对多核糖体进行定量。Ribo Mega-SEC 被证明是一种高效、方便且高度可重复的方法，可用于研究功能翻译复合物。我们表明，Ribo Mega-SEC 很容易与基于 MS 的高通量蛋白质组学结合使用，以鉴定与多核糖体和核糖体亚基相关的蛋白质。它还便于为电子显微镜和结构研究分离复合物。
