We also utilized this procedure followed by TRIzol RNA extraction directly on the beads in order to identify co-purifying RNA with each protein of interest. Beads first underwent three washes in the Lysis Buffer as mentioned previously and then resuspended in 250 μl of Molecular Biology Grade RNAse-free water (Corning). 10 μl of the bead-water mixture was taken for immunoblotting analysis where the beads were mixed with 2X Laemmeli Sample Buffer (Bio-Rad) supplemented with DTT and boiled at 95 °C for five minutes prior to loading onto a BOLT 4–12% Bis-Tris Plus gel (Life Technologies). RNA extraction followed TRIzol LS RNA extraction protocol (Invitrogen). RNA was resuspended in 5 μl of RNAse-free water and loaded onto a 10% polyacrylamide, 7 M urea gel. The gel was then stained with SYBR Gold nucleic acid stain (Invitrogen) to visualize RNA.
