Isolation and Quantification of S. aureus Ribosomes

Crude ribosomes were isolated from S. aureus by cryo-milling methods in buffer A (20 mM HEPES [pH 7.5], 14 mM magnesium acetate [MgOAc₂], 100 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM dithiothreitol [DTT]) (27 (link), 43 (link)). Five absorbance units (A₂₆₀) of ribosomes were layered on a 5% to 30% sucrose gradient that was prepared on a BioComp Gradient Master. The samples were centrifuged at 210,000 × g at 4°C in a SW41 rotor in a Beckman Coulter Optima XPN-100 ultracentrifuge for 3 h. Fractionation was performed using a Brandel fractionation system equipped with a UA-6 UV detector. To quantitate the abundance of total ribosome particles relative to that of the single Δhpf mutant, the boundaries of ribosomal peaks were manually selected from the trough between the peaks. The total area under a peak was calculated by ImageJ and divided to obtain the ratio. When immunoblotting was needed, ∼200 μl per fraction was collected and subjected to final 10% trichloroacetic acid precipitation. The pellets were washed with cold acetone once, resuspended in 50 mM Tris base containing Laemmli sample buffer, and resolved by 4% to 20% TGX SDS-PAGE (Bio-Rad).