Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom
Mark Larance
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
Dylan J Harney
Charles Perkins Centre, School of Life and Environmental Sciences, University of Sydney, Sydney, Australia
Ramasubramanian Sundaramoorthy
Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom
Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom; Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
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.
