Table 1.
ETS enzyme assay reaction conditions for both 37°C and 10°C
| Enzyme | Assay Temperature, °C | Mitochondrial Sample, μg of Total Protein | Reaction Conditions | Absorbance Wavelength, nm |
|---|---|---|---|---|
| Complex I | 37 | 10 | 25 mM K2HPO4 (pH 7.2) | 340 |
| 10 | 50 | 2 μg/ml antimycin A 2 mM KCN 2.5 mg/ml BSA 0.2 mM NADH |
||
| Complex II | 37 | 5 | 25 mM K2HPO4 (pH 7.2) | 600 |
| 10 | 50 | 2 μg/ml rotenone 2 μg/ml antimycin A 2 mM KCN 20 mM succinate 50 μM dichlorophenolindophenol 0.1 mM ubiquinone1 |
||
| Complex III | 37 | 1 | 25 mM K2HPO4 (pH 7.2) | 550 |
| 10 | 10 | 2 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 IV | 37 | 1 | 25 mM K2HPO4 (pH 7.2) | 550 |
| 10 | 5 | 5 mM MgCl2 2.5 mg/ml BSA 0.6 mM lauryl maltoside 50 μM reduced cytochrome c |
||
| Complex V | 37 | 3 | 5 mM ATP | 340 |
| 10 | 40 | 1 mM phosphoenolpyruvate 0.2 mM NADH 1 U/ml pyruvate kinase 1 U/ml lactate dehydrogenase |