Sw40 ti swinging bucket rotor

Exosomes from 500 µL serum were also isolated by UC method. Briefly, the serum was transferred into 12.5 mL ultracentrifuge tube (Beckman). The tube then was filled with PBS (HyClone, SH30256.01) followed by ultracentrifugation twice for 70 min at 100,000 g, 4 °C in an SW 40Ti swinging-bucket rotor (Beckman). The supernatant was discarded and the exosome pellet was used for subsequent experiments.

In vitro translation reactions (4 × 500 μl) were loaded onto 500 μl sucrose cushion (750 mM sucrose) in Buffer C (50 mM HEPES, 250 mM KOAc, 10 mM MgOAc, 0.1% DDM, 1/1,000 complete protease inhibitor (Roche), 0.2 U ml⁻¹ RNasin, pH 7.2 at 4 °C) and centrifuged for 150 min (45,000 r.p.m., 4 °C) in a Beckman Coulter TLA 120.2 fixed-angle rotor. Pellets were resuspended in 4 × 300 μl 250 mM sucrose in ice cold buffer C and loaded onto a Talon metal affinity chromatography column (750 μl resin) pre-equilibrated in 10 ml buffer C containing 10 μg μl⁻¹ bulk tRNA. The column was washed with 25 ml buffer C until no significant absorption (A₂₆₀) could be detected in the wash fractions. The MifM-SRC, bound to the Talon matrix by MifM's N-terminal 8 × His-tag, was eluted in 4 × 500 μl buffer C containing 150 mM Imidazole. The eluate was loaded onto 10–40% sucrose gradients (prepared with buffer C) and centrifuged for 13 h in a Beckman coulter SW40 Ti swinging bucket rotor (20,000 r.p.m., 4 °C). Gradients were separated on a Biocomp Gradient Station ip and fractions containing 70S ribosomal particles were collected and pelleted for 3 h in a Beckman Coulter TLA 120.2 fixed-angle rotor (45,000 r.p.m., 4 °C). The MifM-SRC pellet was resuspended in 28 μl 30 mM sucrose in buffer C, on ice (69 OD ml⁻¹), aliquoted and snap-frozen. Samples were further analysed by SDS–PAGE and western blotting.

A sample containing ~300 μg of PBS was loaded onto sucrose step gradient prepared by layering from bottom upward: 2 mL of 2.0 M, 3 mL of 1.0 M, 2.5 mL of 0.75 M, 2.5 mL of 0.5 M, and 2 mL of 0.25 M sucrose solutions in 0.8 M PB, pH 7.0, in 14 mL Ultra-Clear centrifuge tubes (Beckman, cat. 344060). The resulting gradients were centrifuged in a Beckman SW 40 Ti Swinging-Bucket Rotor at 220,000 g (40,000 rpm) for 18 h at 20 °C. Blue-colored fractions were collected from the sucrose gradients using a syringe. The sucrose in each fraction was diluted with 5 vol of PB before proteins in each fraction were precipitated by adding ammonium sulfate to 50% (w/v) (Safaei et al., 2019 (link); Su et al., 1992 (link)). Resulting pellet was resuspended in 15 μL of PB and used for analysis on SDS-PAGE gel.