| Assay Method Information | |
| | In Vitro Enzyme Activity and Drug Binding |
| Description: | Ub-charging of E2 enzyme was performed for 90 min at 30° C. in a reaction containing 0.1 μM GST-E1, 3.3 μM E2/UbcH7, 10 μM FLAG-Ub and 0.188 μM PINK1 (all R&D systems). In a separate reaction 0.75 μM Parkin (Ubiquigent) was preincubated with 20 μM compound 4 (or equal volume of DMSO). Both reactions were diluted in Ub buffer (final concentration: 20 mM HEPES pH 7.2, 10 mM MgCl2, 0.1 mM EGTA, 500 μM TCEP and 10% ATP regeneration system (20 mM HEPES pH 7.6, 10 mM ATP, 300 mM phosphocreatine, 10 mM MgCl2, 10% glycerol, 1.5 mg/mL creatine phosphokinase (all Sigma Aldrich))). 1 U Apyrase (Sigma) was added per 9 μL reaction and both reactions were combined and incubated for an additional 30 min. 100 μL of 1×LDS buffer was added per 10 μL reaction and samples were split for −/+DTT (20 mM final). |
| Affinity data for this assay | |
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