Isolation of mitochondria

Mitochondria were isolated from human fibroblasts following QproteomeTM Mitochondria isolation kit manual (Qiagen, Cat.37612). In brief, cell pellets were harvested and washed once using 0.9% NaCl, then cells were lysed in buffer and the lysate was centrifuged at 1000 g for 10 min. The supernatant was saved as the cytosolic protein sample. Cell disruption was carried out using a 1 ml syringe with a blunt-ended needle in disruption buffer (provided in the kit) times¹⁰. The lysate supernatants were saved after centrifugation at 1000 g for 10 min. The pellet disruption step was repeated and the supernatants were combined. The mitochondrial pellets were collected after supernatants were centrifuged at 6000 g for 10 min. After washing with storage buffer and centrifuging at 6000 g for 20 min, the mitochondrial pellets were resuspended in lysis buffer as the mitochondrial protein sample.