Ua 6 uv vis detector machine

Polysome isolation was performed as described previously.^{40 (link)} In brief, cells were incubated with 100 μg/ml cycloheximide for 3 min prior harvesting. Cell pellets were lysed with the polysome lysis buffer (0.3M NaCl; 15 mM MgCl2; 15 mM Tris-HCl, pH 7.5) containing 1% v/v Triton-X. The nuclei were precipitated by centrifugation at 13000 rpm (4 °C, 1 min), and the supernatant was loaded onto the top of the 10–50% linear sucrose density gradients. Next, the samples were separated by centrifugation in Sorvall WX Ultra Series Centrifuge (Thermo Fisher Scientific, Waltham, MA, USA) at 38 000 rpm for 2 h at 4 °C. After centrifugation, the samples were fractionated and absorbance at 254 nm was monitored using UA-6 UV/VIS Detector machine (Teledyne Isco, Lincoln, NE, USA). The samples were collected into 12 fractions 1 ml each. Proteins were precipitated using 20% (w/v) trichloroacetic acid and analyzed by immunoblotting.