Supercomplexes, COX and Cox1p intermediates in different COX mutants. Mitochondria of the parental strain W303-1A expressing untagged Cox1p, of MRS/COX1-HAC and of cox4, cox14, coa3 and coa1 mutants, each harbouring the COX1-HAC fusion gene were extracted with digitonin and fractionated as described in Materials and Methods. The fraction eluted from the protein C antibody beads with EDTA was separated by BN-PAGE on 4–13% polyacrylamide gel, transferred to a PVDF membrane and probed with a monoclonal antibody against the HA tag followed with peroxidase coupled anti-mouse antibody. Cox1p-HAC was detected with the Super Signal chemiluminescent substrate kit (Pierce, Rockford, IL).