Express protein labeling mix easy tag

Mitochondria were isolated from third instar larvae and in organello transcription assays were performed as previously described [10 (link),23 (link),36 (link)]. In brief, 200 μg of fresh mitochondria were incubated for 45 min in transcription buffer (30 μCi [³²P]-UTP, 25 mM sucrose, 75 mM sorbitol, 100 mM KCl, 10 mM K₂HPO₄, 50 μM EDTA, 5 mM MgCl₂, 1 mM ADP, 10 mM glutamate, 2.5 mM malate, 10 mM Tris-HCl pH 7.4 and 5% (w/v) BSA), followed by RNA extraction, separation on a 1% MOPS-formaldehyde agarose gel and transferring to Hybond-N+ membranes (GE Healthcare). Mitochondrial de novo translation in isolated mitochondria was assayed as previously described [10 (link),23 (link),36 (link)], using EXPRESS protein labeling mix easy-tag (Perkin Elmer). Equal amounts of total mitochondrial protein were separated on 17% SDS-PAGE gels, followed by staining with 1 g/L Coomassie Brilliant Blue in a 20% ethanol, 10% acetic acid solution. Gels were then destained, dried and exposed to a PhosphorImager screen to visualise the mitochondrial translation products.

Mitochondria were isolated from third instar larvae and in organello transcription assays were performed as previously described (34 (link)). In brief, 200 μg of fresh mitochondria were incubated for 45 min in transcription buffer (30 μCi [³²P]-UTP, 25 mM sucrose, 75 mM sorbitol, 100 mM KCl, 10 mM K₂HPO₄, 50 μM EDTA, 5 mM MgCl₂, 1 mM ADP, 10 mM glutamate, 2.5 mM malate, 10 mM Tris- HCl pH 7.4 and 5% (w/v) BSA), followed by RNA extraction, separation on a 1% formaldehyde agarose gel and transferring to Hybond-N+ membranes (GE Healthcare). Mitochondrial de novo translation in isolated mitochondria was assayed as previously described (34 (link)), using EXPRESS protein labeling mix easy-tag (Perkin Elmer). Equal amounts of total mitochondrial protein were separated on 15% SDS-PAGE gels, followed by staining with 1 g/l Coomassie Brilliant Blue in a 20% ethanol, 10% acetic acid solution. Gels were then destained, dried and exposed to a PhosphorImager screen to visualize the mitochondrial translation products.