Model em 1 econo uv monitor

SDGC experiments were performed according to a modified protocol from Beckert et al. (25 (link)). Cells were grown in MOPS MM as described above and 10–50 ml harvested at indicated time points by centrifugation at 5000 × g at 4°C for 15 min. The cell pellets were frozen in liquid nitrogen and stored at −80°C until further treatment. Cells were re-suspended in 500 μl ice-cold lysis buffer (25 mM HEPES, pH 7.5; 100 mM KOAc; 15 mM Mg(OAc)₂; 1 mM DTT), added to an equal volume of 0.5 mm zirconia/silica beads (BioSpec) and lysed by vortexing in 5 cycles of 1 min vortexing and 1 min on ice. The lysate was cleared by centrifugation at 12 000 × g at 4°C for 5 min. The approximate yield was determined by measuring A₂₆₀ with a NanoVue™ Plus Spectrophotometer (GE Healthcare). The lysate was diluted to 20 A₂₆₀ units in 300 μl ice-cold lysis buffer and layered onto a 5–20% sucrose gradient (25 mM Hepes, pH 7.5; 100 mM KOAc; 15 mM Mg(OAc)₂; 1mM DTT; 0.01% n-dodecyl-d-maltoside; 5–20% sucrose) prepared using a Gradient Master (BioComp). Ribosomal particles where separated by centrifugation at 37 000 rpm/209 627 g at 4°C for 2 h in a Thermo Sorvall WX90 ultracentrifuge with a TH-641 rotor. Obtained gradients were fractionated with a Gradient Fractionator (BioComp) and analysed with a Model EM-1 Econo UV monitor (Biorad) UV detection system at 254 nm.