Ribosomal Protein Sedimentation Analysis

The sedimentation properties of GTPBP8 and the ribosomal proteins in sucrose gradients were analyzed as described before [36 (link)]. Cells or mitochondria were lysed in 1% DDM lysis buffer (50 mM Tris, pH 7.2, 10 mM Mg(Ac)₂, 40 mM NH₄Cl, 100 mM KCl, 1% DDM, 1 mM PMSF, 6 µl/mL Chloramphenicol, and 1 mM ATP) for 20 min on ice, then centrifuged at 20,000 ×g for 20 min at 4 °C. A supernatant containing 900 g of total protein was loaded onto a 16 mL linear 10–30% sucrose gradient (50 mM Tris, pH 7.2, 10 mM Mg(Ac)₂, 40 mM NH₄Cl, 100 mM KCl, and 1 mM PMSF) and centrifuged for 15 h at 4 °C with 74,400 ×g (SW 32.1 Ti; Beckman Coulter). 24 equal volume fractions were collected from the top and subjected to TCA precipitation. Samples were separated by SDS-PAGE for subsequent immunoblotting. For RNaseA treatments, RNaseA (ThermoFisher Scientific) was added to the cell lysate at a final concentration of 600 U/mL at the stage of protein sample preparation, and sucrose gradients were prepared as described.