Yoshikawa Harunori, Sundaramoorthy Ramasubramanian, Mariyappa Daniel, Jiang Hao, Lamond Angus I
Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.
Division of Cell Signalling, Fujii Memorial Institute of Medical Sciences, Institute of Advanced Medical Sciences, Tokushima University, Tokushima, Japan.
Bio Protoc. 2021 Aug 5;11(15):e4106. doi: 10.21769/BioProtoc.4106.
Polysome profile analysis is a popular method for separating polysomes and ribosomal subunits and is typically achieved using a sucrose density gradient (SDG). This has remained the gold standard method since ribosomes were first discovered; however, this method is time-consuming and requires multiple steps from making the gradient and long ultracentrifugation to collecting and analyzing the fractions. Each of these steps in the SDG workflow can introduce potential technical variation that affects the reproducibility of gradient profiles between samples. To address these limitations, we have developed a flexible, alternative approach for analyzing polysomes and ribosomal subunits based on size-exclusion chromatography (SEC), termed 'Ribo Mega-SEC.' In comparison with the SDG method, Ribo Mega-SEC involves a single step using ultra-high-performance liquid chromatography (uHPLC). The entire workflow, from injecting the lysate to collecting the fractions, can be performed in as little as 15 min, with high reproducibility. By varying the pore size of the SEC column, polysomes and ribosomal subunits can be separated using extracts from either human or mouse cultured cell lines or from tissue samples, embryos, or budding yeast. The resulting separated fractions are suitable for analysis using a wide range of subsequent analytical techniques including mass spectrometry (MS)-based proteomics, RNA-Seq, electron microscopy (EM), and multiple biochemical assays.
多核糖体谱分析是一种分离多核糖体和核糖体亚基的常用方法，通常使用蔗糖密度梯度（SDG）来实现。自核糖体首次被发现以来，这一直是金标准方法；然而，该方法耗时且需要多个步骤，从制备梯度、长时间超速离心到收集和分析各组分。SDG工作流程中的每一步都可能引入潜在的技术差异，从而影响样品间梯度图谱的重现性。为解决这些局限性，我们开发了一种基于尺寸排阻色谱（SEC）的灵活的、用于分析多核糖体和核糖体亚基的替代方法，称为“核糖体大尺寸排阻色谱法（Ribo Mega-SEC）”。与SDG方法相比，核糖体大尺寸排阻色谱法只需使用超高效液相色谱（uHPLC）进行一步操作。从注入裂解物到收集各组分的整个工作流程最短可在15分钟内完成，且重现性高。通过改变SEC柱的孔径，可以使用来自人或小鼠培养细胞系或组织样品、胚胎或芽殖酵母的提取物分离多核糖体和核糖体亚基。所得的分离组分适用于使用多种后续分析技术进行分析，包括基于质谱（MS）的蛋白质组学、RNA测序、电子显微镜（EM）以及多种生化测定。
