Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis.In this assay, m RNA/ribosome complexes are isolated from eukaryotic cells.A sucrose density gradient separates m RNAs bound to multiple ribosomes known as polyribosomes from m RNAs bound to a single ribosome or monosome.
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Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination.
Examination of the m RNAs present in the polyribosome fractions can reveal whether the cohort of individual m RNAs being translated changes with experimental conditions.
In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient.
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We recommend downloading the newest version of Flash here, but we support all versions 10 and above. This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells.Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed.As such, this method is the gold standard for examining the regulation of translation.Date Published: 6/15/2010, Issue 40; doi: 10.3791/1948 Keywords: Cellular Biology, Issue 40, translation, ribosome, polyribosome, gradient, fractionation Protein synthesis is a complex cellular process that is regulated at many levels.For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal.Alternatively, translation of individual m RNAs can be regulated by m RNA localization or the presence of cognate micro RNAs.