Mitochondrial Complex Isolation for BN-PAGE

Mitochondria derived from tissues were permeabilized with either 8 mg digitonin/mg protein (heart) or 3 mg digitonin/mg protein (gastrocnemius). Digitonin incubation was performed on ice for 5 min and then centrifuged at 20,000 × g for 30 min as previously described (Acin‐Perez et al, 2008 (link), 2020a (link)). Cells preparations for BN‐PAGE were performed as described (Fernandez‐Vizarra & Zeviani, 2021 (link)). Supernatant containing mitochondrial complexes and super complexes were mixed with Blue Native sample buffer (5% Blue G dye in 1 M 6‐amiohexanoic acid), loaded, and run on a 3–12% native precast gel (Invitrogen). Gels were run till the blue front ran out of the gel and gel was transferred to PVDF membranes.