Sucrose Density Gradient Fractionation

Ten sucrose solutions from 50% to 5% (w/v) sucrose were prepared in 10 mM Tris (pH 7.5), 1 mM EDTA (pH 8) and 100 mM NaCl as previously described^{20 (link)}. First, 1 mL of the 50% sucrose solution was added to the bottom of the tube (Beckman Coulter, 344059) and flash frozen in liquid nitrogen. Then each sucrose solution was layered on top of the previous solution and frozen prior to addition of the next layer with the 5% sucrose solution on top of the tube. The sucrose density gradients were stored at −20 °C and thawed slowly on ice before adding protein lysates.
The Control and RNase samples were carefully overlaid on top of the thawed sucrose gradients avoiding any disturbance. For each condition and replicate, 2 to 2.5 mg of proteins were loaded onto the sucrose gradients. Ultracentrifugation was performed in Beckman L8-70M Ultracentrifuge equipped with a SW 41 Ti Swinging-Bucket Rotor (Beckman Coulter, 331362) at 110,000 x g for 18 h at 4 °C. After centrifugation, 25 fractions (~440 μL each) were carefully transferred by pipetting into fresh 1.5 mL tubes. Fraction 1 corresponded to the top of the tube and fraction 25 to the bottom. The different fractions were stored at −80 °C for western blot analysis or precipitated with 20% trichloroacetic acid (TCA) for mass spectrometry.