Easy tag express protein labeling mix 35s

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).

The pulse labeling of mitochondrial protein synthesis was performed essentially as described^{69 (link)}. WT or OSGEPL1 KO HEK293T cells (2 × 10⁶ cells) were cultured at 37 °C for 15 min in 6 mL of L-glutamine/L-cysteine-free DMEM (21013-024, Gibco) containing 10% dialyzed FBS (Gibco), 2 mM L-glutamine (Sigma), and 1 mM sodium pyruvate (Gibco), followed by addition of 77 μg/mL emetine (Sigma) and incubation for 5 min. Then, Easy Tag Express Protein Labeling Mix [³⁵S] (NEG772007MC, Perkin Elmer) was added and the sample incubated at 37 °C for 1 h to specifically label mitochondrial proteins. Cell lysates (100 μg total proteins) were resolved by Tricine-SDS-PAGE (16.5%) and the gel was CBB-stained and dried on a gel drier (AE-3750 RapiDry, ATTO). Radiolabeled mitochondrial protein products were visualized by exposure to an imaging plate (BAS-MS2040, Fujifilm) on a fluorimager (FLA-7000, Fujifilm).