Fig. 1

Purity and function of liver mitochondria comparing isolation with differential centrifugation versus a Percoll gradient. a Western blot. Lanes 1–4 are liver mitochondria isolated by differential centrifugation. Lanes 5–8 are liver mitochondria isolated using a Percoll gradient. Protein was loaded at 25 μg/lane. COX IV antibody [20E8] (Abcam) was used to detect mitochondrial protein. GAPDH, an enzyme expressed in the cytosol, demonstrates minimal differences in cytosolic contamination among the preparations. b Densitometry analysis of the immunoblots shows there is no difference in the amount of COX IV protein in the mitochondrial preparations resulting from these two methodologies. c Respiration. Liver mitochondria were isolated by either differential centrifugation (open bars) or with Percoll (closed bars) and then tested for respiratory capacity. Mitochondrial respiration was measured using the complex I substrates glutamate and malate. State 1 respiration was recorded without substrates, subsequently state 2 respiration was recorded after the addition of substrates, and finally ADP (0.5 mM final concentration) was added in order to induce state 3 respiration. n = 6 for both mitochondrial preparations and for all three respiratory states. Respiratory rates were comparable for the two mitochondria isolated by the two methods without significant differences