Figure 2. Reporter based enrichment assay for ribosome associated trans-translation factors.

[A] Schematic representation of a ribosome enrichment experiment. Total ribosomes can be obtained via any of the methods described in the main text. Isolated total ribosomes comprise a mixture of those translating normal cellular mRNA, or the reporter nonstop mRNAs encoding a His6 epitope tag. Stalled and rescued trans-translating ribosomes can be separated from the normal ribosome pool by using a suitable affinity column. The figure depicts enrichment of ribosomes translating a nonstop mRNA encoding an N-terminal His6-tagged reporter protein using a Ni²⁺-NTA affinity column. Enriched ribosomes are subjected to western blot analysis, using antibodies specific to the protein of interest. [B] Representative western blot showing enrichment of SmpB on ribosomes translating λ-cI-nonstop mRNA. Cushion purified total ribosomes were segregated into normal and stalled or trans-translating ribosomes using Ni²⁺-NTA column chromatography. Total and eluted enriched ribosomes were normalized by A₂₆₀ and resolved by electrophoresis on a 10% SDS-acrylamide gel. The gel was used for electrophoretic transfer and western blot analysis using anti-SmpB antibodies. SmpB was enriched on captured ribosomes translating the reporter nonstop mRNA.