After electrophoresis, gels were further processed for either western blotting or In Gel Activity IGA assays.
In order to analyze the assembly status of respiratory complexes and supercomplexes, proteins separated by BN were transferred onto PVDF membranes and probed with specific antibodies that recognize the different respiratory complexes (anti-NDUFA9, anti-SDHA, anti-Uqcrc1 and anti-COI for complexes I, II, III, IV respectively, all of them from Invitrogen, and anti-β-F1-ATPase, for complex V, kindly gifted by Dr. J.M. Cuezva). Then, they were incubated with their corresponding secondary antibody, tagged with peroxidase (ThermoFisher Scientific, Waltham, MA, USA). Finally, protein levels were evaluated by immunoblot, using an AmershamTM Imager 600 to document the signal.
Complex I IGA assays were performed after BN-PAGE by incubating the gels at room temperature in the presence of 0.1 M Tris-HCl pH 7.4, 0.14 mM NADH and 1 mg/mL of nitro-blue tetrazolium (NBT) as previously described [80 (link)]. Complex V IGA staining was performed by incubating the gels in 34 mM Tris, 270 mM glycine, 14 mM MgSO4, 0.2% Pb(NO3)2 and 8 mM ATP at pH 7.8, as described before [81 (link)]. After incubation, the gels were photographed with a digital camera using as background a white translucid screen for Complex I and Complex IV and a black screen for Complex V [81 (link)].
In order to analyze the assembly status of respiratory complexes and supercomplexes, proteins separated by BN were transferred onto PVDF membranes and probed with specific antibodies that recognize the different respiratory complexes (anti-NDUFA9, anti-SDHA, anti-Uqcrc1 and anti-COI for complexes I, II, III, IV respectively, all of them from Invitrogen, and anti-β-F1-ATPase, for complex V, kindly gifted by Dr. J.M. Cuezva). Then, they were incubated with their corresponding secondary antibody, tagged with peroxidase (ThermoFisher Scientific, Waltham, MA, USA). Finally, protein levels were evaluated by immunoblot, using an AmershamTM Imager 600 to document the signal.
Complex I IGA assays were performed after BN-PAGE by incubating the gels at room temperature in the presence of 0.1 M Tris-HCl pH 7.4, 0.14 mM NADH and 1 mg/mL of nitro-blue tetrazolium (NBT) as previously described [80 (link)]. Complex V IGA staining was performed by incubating the gels in 34 mM Tris, 270 mM glycine, 14 mM MgSO4, 0.2% Pb(NO3)2 and 8 mM ATP at pH 7.8, as described before [81 (link)]. After incubation, the gels were photographed with a digital camera using as background a white translucid screen for Complex I and Complex IV and a black screen for Complex V [81 (link)].