Sucrose cushion for ribosome pelleting

Sucrose cushion fractionation was performed as described previously¹⁵. 50 ml of each yeast culture was grown at 30 °C to an OD₆₀₀ of 0.6. Cycloheximide was added to the culture to a final concentration of 100 μg/ml, to trap translating ribosome-mRNA complexes. The culture was incubated for another 15 min at 30 °C, cells collected by centrifugation and lysed in chilled CSB buffer (300 mM Sorbitol, 20 mM HEPES pH 7.5, 1 mM EGTA, 5 mM MgCl₂, 10 mM KCl, 10% (w/v) Glycerol, 100 μg/ml cycloheximide, 10 u/ml SUPERaseIn RNAse inhibitor and 1 Pierce protease inhibitor cocktail) with glass bead homogenization for 5 × 20 secs in low RNA-binding tubes (Life Technologies). 500 μg of the protein extract dissolved in 500 μl of CSB was gently lowered over a 400 μl of 50% (w/v) sucrose plus CSB buffer without sorbitol and protease inhibitor to form two distinct layers in Beckman thickwall polycarbonate tubes (Beckman). The tubes were centrifuged in an ultracentrifuge (rotor-TLA 120.2, used in Beckman Optima Max-XP centrifuge) for 2.5 h at 4 °C, 70,000 rpm. The position on the tube where the pellet was deposited was noted and the supernatant transferred into a new microfuge tube. The pellet was thoroughly resuspended in 100 μl Novex tricine SDS sample buffer (Invitrogen). To the supernatant fractions, TCA (trichloroacetic acid) was used to precipitate ribosome-free proteins. After acetone washes pellets were resuspended in 100 μl Novex tricine SDS sample buffer (supernatant fraction). 10 μl (10%) of both the pellet and supernatant fractions were analysed by SDS tricine gels and western blotting.