Trans blot semi dry transfer apparatus

20–40 μg of each protein lysate was separated via SDS-PAGE utilizing a 10% polyacrylamide gel. Proteins were transferred to nitrocellulose membrane using the Bio-Rad Trans-Blot semi-dry transfer apparatus. Blocking was performed for 20 min using 5% nonfat dried milk in TBS-0.1% Tween (TBS-T). Primary antibodies were diluted 1:1000 in TBS-T containing 10% Super Block T20 (Thermo Scientific) and incubated with the blot overnight at 4°C. After three washes for 10 min in TBS-T, the blot was incubated with a horseradish peroxidase (HRP) -conjugated secondary antibody at 1:5000, diluted in TBS-T containing 10% Super Block T20. Clarity Western ECL Substrate (Bio-Rad) was used to detect the HRP of the secondary antibody. Imaging was performed on the ChemiDoc MP imaging system and Image Lab software (Bio-Rad). These experiments were conducted in at least three independent replications, and the image is a representative sample.

Total cell lysates were prepared using radioimmunoprecipitation assay buffer (Sigma-Aldrich, St. Louis, MO, USA) containing protease inhibitors (Roche, Bassel, Switzerland). Protein concentrations were determined using a BCA protein assay kit (Beyotime, Shanghai, China). Equal quantities of protein were separated by SDS-PAGE then transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA) using a Trans-Blot Semi-Dry Transfer apparatus (Bio-Rad, Hercules, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Blots were probed with the relevant antibodies, and proteins were detected using enhanced chemiluminescence reagent (Bio-Rad, Hercules, CA, USA).

From each selected sucrose gradient fraction (Fig. 1b,c), 10 µL were mixed with equal volume of 2× loading buffer (120 mM Tris-HCl pH 7.5 (25 °C), 40% glycerol, 0.09% bromophenol blue, 20 mM DTT). As control samples, we used RsfS protein (0.1 µg), 70S ribosome (1.1 µg) fraction from sucrose gradient at 3 mM Mg²⁺ (Fig. 1a) and 70S + 5X RsfS mixture (containing 1.5 µg 70S and 0.35 µg RsfS) before centrifugation in sucrose gradient. Proteins were separated on 15% polyacrylamide gel according to Leammli electrophoresis protocol^{47 (link)}. The proteins were transferred to a 0.45 µm nitrocellulose membrane at 10–11 V for 45 min at room temperature using the Trans-Blot Semi-Dry Transfer apparatus (BioRad). After incubation with 5% nonfat milk in TBST (10 mM Tris, pH 8.0 150 mM NaCl, 0.5% Tween 20) for 60 min at 37 °C, the membrane was washed once with TBST and incubated with monoclonal anti-polyhistidine-peroxidase clone HIS-1 antibody (Sigma-Aldrich) at 1:2000 ratio at 37 °C for 1 h. Membranes were washed four times for 10 min and developed with Pierce™ ECL western blotting substrate, and detected by Thermo SuperSignal™ West Femto Maximum Sensitivity Substrate and the ECL system (Amer Biosciences) according to the manufacturer’s protocols.