Depletion and reconstitution of 5S RNP complexes in early pre60S ribosomes

We purified pre60S ribosomes from yeast using Nsa1-FtpA as bait in Tandem Affinity Purification experiments. We tested the depletion of RPF2, using a Galactose-depletion system, to obtain ribosomal particles depleted of 5S RNP complexes. Sample A1: Control group (wild type strain) grown in YPGal; Sample A2: Control group (wild type strain) grown in YPGlu; Sample A3: Control group (Gal::HA-RPF2 strain) grown in YPGal; and Sample A4: Test group (Gal::HA-RPF2) shifted from YPGal to YPGlu for 6 hours to achieve 5S RNP depletion. Sample N1: Purified pre-60S Nsa1-associated with 5S RNP depleted after Sucrose gradient separation; Sample N2: Purified pre-60S Nsa1-associated with 5S RNP reconstituted after Sucrose gradient separation.