Alkaline phosphatase conjugated anti mouse or anti rabbit secondary antibodies

Total yeast protein extracts were prepared and analyzed by western blotting according to standard procedures (45). Commercial (anti-GFP, Roche; anti-Myc, Sigma-Aldrich; anti-Nop2, Thermo Fisher Scientific) and specific primary antibodies against r-proteins and AFs were used. Secondary goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (Bio-Rad), or alkaline-phosphatase-conjugated anti-mouse or anti-rabbit secondary antibodies (Promega) were used. Proteins were detected using a chemiluminescence detection kit (Super-signal West Pico, Pierce) and a ChemiDoc MP™ system (Bio-Rad). Alternatively, colorimetric detection was performed using NBT and BCIP (Promega).

Proteins from whole-cell extracts or from purified pre-ribosomes were separated on SDS-PAGE gels, electroblotted to the Amersham Protran supported 0.45 µm NC membrane (GE Healthcare Life Sciences), and assayed by western blot analysis. To conserve antiserum by using a lower volume of blotting buffer and to enable detection of multiple proteins on one blot, nitrocellulose membranes were cut into smaller sections based on the known mobility of the different proteins. As Nog2 co-migrates with IgG on 4–20% Tris-Glycine Novex gels, NuPage 4–12% Bis-Tris gels (Thermo Fisher Scientific) were used to assay Nog2 protein by western blotting. TAP-tagged proteins were detected using alkaline phosphatase conjugated to IgG (Pierce). HA-tagged proteins were identified with mouse monoclonal antibody 12CA5 (Thermo Fisher Scientific) and Myc-tagged proteins with 9e10 antibody (Sigma-Aldrich). Otherwise, antibodies specific for r-proteins or AFs were used. Alkaline-phosphatase-conjugated anti-mouse or anti-rabbit secondary antibodies (Promega) were used and colorimetric detection was performed using NBT and BCIP (Promega).