Tla 120.2 fixed angle rotor

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.

500 μL in vitro translation reaction was loaded onto 10–50% sucrose gradient prepared with Buffer C (25 mM pH 7.2 HEPES-KOH, 100 mM KOAc, 10 mM Mg(OAc)₂, 0.01% DDM, 1/1,000 complete protease inhibitor (Roche, Germany), 0.2 U/mL RNase, 2 mM 2-mercaptoethanol) and centrifuged for 3 hr in a Beckman coulter SW40 Ti swinging bucket rotor with 35,000 r.p.m. at 4°C. Gradients were separated on a Biocomp Gradient Station and fractions containing 70S ribosomal particles were collected and loaded onto a Talon metal affinity chromatography column (1.5 ml resin) pre-equilibrated in 10 mL buffer C containing 10 μg/mL bulk tRNA. The column was washed with 25 ml buffer C until no significant absorption (OD₂₆₀) could be detected in the wash fractions. The VemP-SRC, bound to the Talon matrix by the VemP N-terminal His-tag, was eluted in 750 μL buffer C containing 150 mM imidazole. The elution was pelleted for 4 hr 20 min in a Beckman Coulter TLA 120.2 fixed-angle rotor with 51,000 r.p.m. at 4°C. 15.6 pmol VemP-SRC pellet was resuspended in ice-cold buffer C without DDM, aliquoted and snap-frozen.

Duplicate samples of freshly-collected EDTA-plasma were used to separate the three major lipoprotein classes by sequential, isopycnic centrifugation in a TLA-120.2 fixed angle rotor (Beckman, Fullerton, CA), as described previously [55 (link)]. Density (r) adjustments were made with stock solutions of KBr in 0.15 M NaCl and four fractions were obtained: very-low density lipoproteins (VLDL; r <1.006 g/L), low-density lipoproteins (LDL; r: 1.006–1.063 g/L), high-density lipoproteins (HDL; r: 1.063–1.21 g/L), and very-high-density lipoproteins (r>1.21 g/L, which also include bulk plasma proteins). Each fraction was collected, extensively dialyzed against 150 mM NaCl/ 0.24 mM EDTA pH 7.4 at 4°C and filtered through 0.22 μm-pore size filters (Millipore S.A., St Quentin, France).