Global Assessment of Protein Translation in Mammalian Cells Using Polysome Fractionation

Abstract

A character of active protein translation is formation of multiple ribosomes, or polysomes, on translating mRNAs. Polysome intensity reflects global cellular translation activity and can be assessed after biochemical fractionations of polysomes. Polysome fractionation begins with immobilizing ribosomes on mRNAs using inhibitors of translation elongation, for example, cycloheximide. Nuclei-free cell lysates are then isolated and layered on the top of a sucrose gradient for ultracentrifugation to separate ribosomal subunits, monosome, and multiple fractions of polysomes by their different sedimentation rates along the sucrose gradient. A density gradient fractionation system including a spectrophotometer reads the RNA absorbance of the flowed gradient and generates the fractions. These fractions can be subjected to further RNA and protein analyses, for example, polysome profiling and mass spectrometry. Here, we present a detailed protocol of polysome fractionation for mammalian cells.

Keywords: Cycloheximide; Fractionation; Polysome profiling; Sucrose gradient; Translatome.