<em>Mycobacterium smegmatis</em> Ribosome Purification, Co-sedimentation, and Subunit Association Assay

The ribosome, a complex macromolecular machine, plays a vital role in cellular translation. To investigate its structure and conduct in vitro experiments, isolating the ribosomes from cells is the first step. While isolating ribosomes from bacterial cells is routine, obtaining them from mycobacteria proves challenging due to the protective mycolic acid layer, which hinders cell lysis. In this study, we present a straightforward and efficient protocol for isolating ribosomes from <i>Mycobacterium smegmatis</i>. Additionally, we introduce a co-sedimentation assay using density gradient ultracentrifugation, providing a simple yet powerful method for studying ribosome-protein interactions. The re-association assay also offers a practical approach for obtaining tRNA-free 70S ribosomes and evaluating the anti-association properties of potential ligands. While these assays are commonly used, our protocol stands out for its simplicity, requiring limited specialized instruments. These methods can also be scaled up or down per requirement. By employing sonication for cell rupture and utilizing basic lab equipment for ultracentrifugation-based assays, our method greatly simplifies ribosome isolation and related research. Key features • Cellular ribosome isolation from <i>Mycobacterium smegmatis</i> using an optimized sonication cycle. • Detection of ribosome-protein interactions through density gradient ultracentrifugation. • Simple steps to obtain tRNA-free 70S ribosomes that are crucial for several biochemical experiments. • Screening of proteins or ligands for their ribosomal anti-association activity.