Coupled in vitro transcription and translation assays were performed using the TNT Quick Coupled Transcription/Translation system (Promega) as described previously (16 (link)). Reactions contained 10 μL lysate with 250 ng of pcDNA T7 expression plasmid and 0.5 μL [35S] methionine/cysteine (PerkinElmer). Reactions were incubated at 30°C for 40 min chasing with 2 μL of 50 mg/mL unlabeled methionine/cysteine. Reactions were stopped at 20-min or hourly intervals by the addition of 2× Laemmli buffer. Samples were separated by SDS-PAGE before visualization of radiolabeled products by autoradiography.
For the trans-cleavage assays, the TNT reactions were supplemented with purified FMDV 3Cpro (a kind gift from Dr. Tobias Tuthill [57 (link)]) to the indicated final concentration from dilution of a 1 mM stock, simultaneous to the addition of unlabeled methionine/cysteine. Reactions were stopped at 20-min or hourly intervals by the addition of 2× Laemmli buffer and the 3Cpro-mediated proteolysis of radiolabeled precursor monitored by SDS-PAGE.
For the trans-cleavage assays, the TNT reactions were supplemented with purified FMDV 3Cpro (a kind gift from Dr. Tobias Tuthill [57 (link)]) to the indicated final concentration from dilution of a 1 mM stock, simultaneous to the addition of unlabeled methionine/cysteine. Reactions were stopped at 20-min or hourly intervals by the addition of 2× Laemmli buffer and the 3Cpro-mediated proteolysis of radiolabeled precursor monitored by SDS-PAGE.
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