Mitochondria were isolated using the Qproteome Mitochondria Isolation Kit or another commercially available kit (Supplier S) according to manufacturer’s instructions. Mitochondrial membrane integrity is indicated by formation of fluorescent aggregates following uptake of a carbocyanine dye.
[A] Mitochondria were prepared from 60 mg of liver tissue using the Qproteome Mitochondria Isolation Kit and subjected to 2D-PAGE. Out of 384 spots picked, 259 proteins were identified by MALDI-MS, 219 (85%) of which were predicted to have a mitochondrial localization. Green circles indicate mitochondrial proteins, red circles indicate impurities, the black circle indicates a protein which is probably located in mitochondria and ER. [B] Electron micrographs of mitochondria isolated using the Qproteome Mitochondria Isolation Kit showing intact inner and outer membranes.
Western blots of mitochondrial preparations probed with antibodies specific for organelle-/cell-compartment specific marker proteins. PM: plasma membrane; ER: endoplasmic reticulum.