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| | Inhibition Assay of Cdc7 Activity |
| Description: | The assay consists of the transfer of radioactivity labeled phosphate moiety by the kinase to an acceptor substrate. The resulting 33P-labeled product is separated from unreacted tracer, transferred into a scintillation cocktail and light emitted is measured in a scintillation counter. The inhibition assay of Cdc7/Dbf4 activity is performed according to the following protocol. The MCM2 substrate is trans-phosphorylated by the Cdc7/Dbf4 complex in the presence of ATP traced with γ33-ATP. The reaction is stopped by addition of Dowex resin in the presence of formic acid. Dowex resin particles capture unreacted γ33-ATP and drag it to the bottom of the well while 33P phosphorylated MCM2 substrate remains in solution. The supernatant is collected, transferred into Optiplate plates and the extent of substrate phosphorylation is evaluated by β counting. The inhibition assay of Cdc7/Dbf4 activity was performed in 96 wells plate according to the following protocol. |
| Affinity data for this assay | |
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