Mitochondrial translation product analysis

Mitochondrial translation products were pulse-labeled in vitro with [³⁵S]-(methionine and cysteine) (GE Healthcare). In experiments where the label was chased, MEF cells were incubated for 6 min with 100 μg/ml emetine or 250 μg/ml CAP prior to labeling for 60 min. Labeled cells were then rinsed with an isotonic buffer (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 10 mM KCl and 0.7 mM Na₂HPO₄). After centrifugation at 1150×g for 5 min, cell pellets were resuspended in loading buffer consisting of 93 mM Tris-HCl, pH 6.7, 7.5% glycerol, 1% SDS, 0.25 mg/ml Bromophenol Blue and 3% mercaptoethanol. The total lysate was then subjected to 15% SDS/PAGE for 3 h at 180 V. Gels were examined using a Fujifilm BAS-2500 Bio Imaging Analyzer (Fujifilm, Tokyo, Japan) [3 (link)].