The Lamond laboratory have developed an efficient and highly reproducible new methodology for isolating polysomes and other large subcellular structures, termed ‘Ribo Mega-SEC’, which they have used to study protein translation complexes in cell lines and tissues.
In a new publication by Yoshikawa et. al. (eLife, 2018), the Lamond group, with help from the Owen-Hughes group, report a powerful new approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits, using Size Exclusion Chromatography and uHPLC. This was shown to provide advantages over conventional methods for polysome fractionation using sucrose density gradients (SDG). For example, it is faster and more reproducible than SDG, allowing functional polysomes to be separated in less than 15 min from sample injection to fraction collection, using extracts from either cells, or tissues. Ribo Mega-SEC shows translating ribosomes in human cell lines and mouse liver exist predominantly in polysome complexes. Changes in polysomes were identified upon the cellular response to amino acid starvation. Ribo Mega-SEC was used in combination with high-throughput, MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It was also shown to facilitate isolation of polysomes and other large complexes for electron microscopy and structural studies.