Polysome profiling is an extensible tool for the analysis of bulk protein synthesis, ribosome biogenesis, and the specific steps in translation.

Protein synthesis is an essential and highly regulated cellular process. Here, we demonstrate the versatility of polysome profiling-a methodology traditionally used to assess levels of protein synthesis-to monitor ribosomal integrity and modulation of specific steps in mRNA translation. Using expanded polysome profiling methodologies, we systematically illustrate defects in ribosome biogenesis, translation initiation, and translational elongation in different cellular conditions. We additionally provide instruction for how a modified polysome profiling protocol can be leveraged to identify and characterize the function of factors that regulate protein synthesis. These methodologies are broadly applicable to a range of physiological conditions and human diseases in which ribosome biogenesis or the phases of protein synthesis are distinctly regulated or dysregulated.