Efficient and Rapid Analysis of Polysomes and Ribosomal Subunits in Cells and Tissues Using Ribo Mega-SEC

Harunori Yoshikawa (Lead / Corresponding author), Ramasubramanian Sundaramoorthy, Daniel Mariyappa, Hao Jiang, Angus I. Lamond (Lead / Corresponding author)

    Research output: Contribution to journalArticlepeer-review

    3 Citations (Scopus)
    309 Downloads (Pure)

    Abstract

    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, Drosophila 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.

    Original languageEnglish
    Article numbere4106
    Pages (from-to)1-15
    Number of pages15
    JournalBio-Protocol
    Volume11
    Issue number15
    DOIs
    Publication statusPublished - 5 Aug 2021

    Keywords

    • MRNA translation
    • Polysome
    • Polysome profile
    • Protein synthesis
    • Ribosome
    • Size-exclusion chromatography
    • Sucrose density gradient

    ASJC Scopus subject areas

    • General Biochemistry,Genetics and Molecular Biology
    • General Immunology and Microbiology
    • General Neuroscience
    • Plant Science

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