Abstract: The efficient separation of ribosomal complexes from free ribosomal subunits was achieved by size-exclusion chromatography (SEC) using a high-performance liquid chromatography (HPLC) or AKTA pureTM liquid chromatography system. First, the UV absorption monitoring system and fraction collection system were used to achieve the separation and collection of the eluted fractions; subsequently, the fractions containing the active polysomes, formed by multiple mRNA-bound ribosome complexes during translation, were confirmed by ribosomal protein-specific Western blot and rRNA or poly(A)-specific RNA dot blot analysis. The results showed that both HPLC and AKTA pureTM liquid chromatography systems could efficiently separate actively translated polysomes from ribosomal subunits, and that the AKTA pureTM liquid chromatography system based on a SuperoseTM6 chromatography column achieved higher recovery for the separation of polysomes. The findings suggested that size exclusion chromatography could be used for the analysis and preparation of active ribosomal complexes, providing reliable support for the discovery of novel ribosome-binding factors and the elucidation of new mechanisms of translational regulation under physiological or pathological conditions.

Key words: ribosome, sucrose density gradient centrifugation, size exclusion chromatography, polysome, ribosomal RNA