Table 1.

ETS enzyme assay reaction conditions for both 37°C and 10°C

EnzymeAssay Temperature, °CMitochondrial Sample, μg of Total ProteinReaction ConditionsAbsorbance Wavelength, nm
Complex I371025 mM K2HPO4 (pH 7.2)340
10502 μg/ml antimycin A
2 mM KCN
2.5 mg/ml BSA
0.2 mM NADH
Complex II37525 mM K2HPO4 (pH 7.2)600
10502 μg/ml rotenone
2 μg/ml antimycin A
2 mM KCN
20 mM succinate
50 μM dichlorophenolindophenol
0.1 mM ubiquinone1
Complex III37125 mM K2HPO4 (pH 7.2)550
10102 μg/ml rotenone
2 mM KCN
2.5 mg/ml BSA
0.6 mM n-dodecyl-β-d-maltoside
15 mM oxidized cytochrome c
0.26 mM ubiquinone2
Complex IV37125 mM K2HPO4 (pH 7.2)550
1055 mM MgCl2
2.5 mg/ml BSA
0.6 mM lauryl maltoside
50 μM reduced cytochrome c
Complex V3735 mM ATP340
10401 mM phosphoenolpyruvate
0.2 mM NADH
1 U/ml pyruvate kinase
1 U/ml lactate dehydrogenase