() Translation inhibition assay

Copyright information:Taken from "Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses"Nucleic Acids Research 2007;35(5):1660-1672.Published online 18 Feb 2007PMCID:PMC1865052.© 2007 The Author(s). The potency of TCS variants to inhibit translation was measured by adding 3.7 pM to 370 nM of protein samples to nuclease-untreated rabbit reticulocyte lysate as described in the Materials and methods section. IC, the concentration of TCS required to achieve 50% inhibition, was determined by fitting the data to a four-parameter logistic equation. The values of IC for WT (filled circle), K173A (open square), R174A (open diamond), D176A (open triangle), K177A (open circle), and the triple-alanine variant (filled square) were 0.027 ± 0.001, 0.20 ± 0.04, 0.16 ± 0.02, 0.038 ± 0.004, 0.09 ± 0.01, 0.5 ± 0.1nM, respectively. () Depurination assay of TCS and its variants. After incubation with 10 nM of TCS or its variants, RNA from the rabbit reticulocyte lysate was extracted, treated with aniline and analysed by electrophoresis as described in the Materials and methods section. Depurination of 28S rRNA at A was detected by the ∼450 bp R-fragments (indicated by an arrow). Control lanes, in which RNA samples were not treated with aniline, are labelled with the ‘−’ marks.