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. 2015 Mar 25;10(3):e0122017. doi: 10.1371/journal.pone.0122017

Fig 2. In vitro translation of HaloTag proteins harbouring the arrest sequence.

Fig 2

(A) Halo-L8-SecM133–170 (lane 1), Halo-L17-SecM133–170 (lane 2), Halo-L26-SecM133–170 (lane 3), Halo-pD-L8-SecM133–170 (lane 4) and Halo-SecM1–170 (lane 5) were translated in the presence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added to the reaction mixture at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after 3-min incubation and subjected to NuPAGE. Polypeptides labelled with HaloTag TMR Ligand were detected using Molecular Imager FX. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. (B) Myc-Halo-L8-SecM133–170 (lane 1), myc-Halo-L17-SecM133–170 (lane 2), myc-Halo-L26-SecM133–170 (lane 3), myc-Halo-pD-L8-SecM133–170 (lane 4) and myc-Halo-SecM1–170 (lane 5) were translated in the absence of HaloTag TMR Ligand using the PURExpress ΔRibosome Kit at 37°C for 20 min. Puromycin (1 mg/mL) was added at 0 min, and the reaction mixture was incubated at 37°C for 3 min. Aliquots were withdrawn before the addition of puromycin and after a 3-min incubation and subjected to NuPAGE. Myc-tagged polypeptides were detected by western blotting with anti-c-myc-tag. Black and white arrowheads indicate the translation arrest products (polypeptidyl-tRNA) and released products, respectively. The results shown are representative of three independent experiments with similar results. (C) Fractions of translation arrest products in the absence (left) and the presence of puromycin (right). Filled bars, fluorescence detection using HaloTag TMR Ligand; open bars, detection by western blotting. Error bars represent the standard deviation (SD) of three independent experiments. The asterisk indicates statistical significance as determined by the Student's t-test (p < 0.05).