Protein a agarose

Transcripts encoding versions of AGAL or Prl-AGAL lacking a termination codon were translated in the presence of CRM for 30 min at 32°C. The membrane-targeted RNCs were isolated by centrifugation through a 0.5 M sucrose cushion for 12 min at 70,000 rpm/TLA100.3, and the resulting pellet was resuspended in membrane buffer (MB: 50 mM Hepes, pH 7.4, 100 mM KAc, 5 mM MgAc, and 250 mM sucrose). For Fig. 4 H, crosslinking was performed with 400 μM bismaleimidohexane (Thermo Fisher Scientific) for 15 min at 25°C and quenched with 25 mM 2-mercaptoethanol. For Fig. 5, A and B, crosslinking was carried out with 200 μM disuccinimidyl suberate (Thermo Fisher Scientific) for 30 min at room temperature and quenched 20 mM Tris-HCl, pH 8.0, for 15 min. The cross-linked products were denatured with 1% SDS, 100 mM Tris-HCl, pH 8.0, for 30 min at 55°C, and diluted 10-fold in Triton buffer. The respective antibodies were added and incubated for 1.5 h at 4°C with end-to-end rotation, followed by incubation with protein-A agarose (Repligen) for 1.5 h at 4°C. The beads were washed three times with Triton buffer, eluted with SDS sample buffer, and analyzed after boiling for 5 min, but 55°C/30 min for the Sec61α sample. Samples were treated with RNase A (100 μg/ml) for 15 min at 37°C before analyzing by SDS-PAGE and autoradiography.