Fig. (12) Capture of intact 80S ribosome from HEK-293T cells using RPS9-HT7. A. Overexpressed RPS9-HT7 (or HT7 alone, control) was captured to HaloLink resin and treated with TEV protease to release RPS9 and its interacting partners. The eluted samples were analyzed by SDS-PAGE and silver staining. Mass analysis of the same samples verified the following was present: 31 of 33 40S proteins, 42 of 50 60S proteins, 2 poly-A binding proteins, 1 GNF exchange protein, 9 nuclear ribonucleoproteins, 2 initiation factors, 2 elongation factors, and 2 splicing factors. For a complete list see Table S7. BIn vitro luciferase translation assay showing activity of ribosomes isolated via RPS9-HT7. RPS9-HT7 was transiently expressed in HEK-293T cells stably expressing Fluc mRNA. Ribosomes were isolated via RPS9-HT7 and released using TEV protease. HT7 alone and untransfected cells were processed in the same manner as negative controls. Signal to background calculations indicated the generation of active luciferase from the RPS9- HT7 complex isolation but not from the negative controls. Commercially available native ribosomes, included as a positive control, were also able to generate active luciferase in vitro.