In vitro tagging of truncated ribosomal protein L27 by hybrid tmRNAs in the presence of either E. coli or M. tuberculosis SmpB proteins. (A) Western Blot analysis. Tagging reactions were assembled by addition of circular plasmid pETrpmA-At encoding truncated protein L27, purified SmpB proteins and tmRNAs to the T7 S30 transcription/translation mixture (Promega). After 60 min incubation at 37 oC, tagged proteins were captured on Ni2+-NTA-Sepharose and then fractionated on a 10% SDS-polyacrylamide gel. Tagging was visualized by Western blotting with anti-T7 tag antibodies in ECL-Plex system. L27* denotes tagged ribosomal protein L27. (B) Graphical representation of Typhoon-derived data from the Western blot. Tagging efficiencies of hybrid tmRNA derivatives were normalized and compared to the tagging efficiency of the E. coli tmRNA(H8):SmpB complex. Error bars show the standard deviation of three or more independent experiments.