Pulse-chase experiments using 9.8 Mbq per dish of EASY-TAG EXPRESS Protein labeling mix [35S] (PerkinElmer) and subsequent immunoprecipitation using the anti-insulin monoclonal antibody and protein G-coupled Sepharose beads (GE Healthcare) were performed according to a published procedure (Ninagawa et al., 2014 (link)).
In vitro translation was performed using the TNT Quick Coupled Transcription/Translation Systems (Promega) and the EASY-TAG EXPRESS Protein labeling mix [35S] (PerkinElmer) according to the manufacturer’s instructions. Translated proteins were subjected to immunoprecipitation using the anti-insulin monoclonal antibody to separate them from 35S-methionine and 35S-cysteine. The immunoprecipitates were analyzed using SDS-PAGE (15 or 16% gel). Radiolabeled proteins were visualized using an FLA-3000G FluoroImage analyzer (Fuji Film).
In vitro translation was performed using the TNT Quick Coupled Transcription/Translation Systems (Promega) and the EASY-TAG EXPRESS Protein labeling mix [35S] (PerkinElmer) according to the manufacturer’s instructions. Translated proteins were subjected to immunoprecipitation using the anti-insulin monoclonal antibody to separate them from 35S-methionine and 35S-cysteine. The immunoprecipitates were analyzed using SDS-PAGE (15 or 16% gel). Radiolabeled proteins were visualized using an FLA-3000G FluoroImage analyzer (Fuji Film).
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