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| | Kinase Inhibition Assay |
| Description: | Phosphorylation reactions were monitored using a coupled-enzyme assay in which ADP production was coupled to NADH oxidation by pyruvate kinase and lactate dehydrogenase. The assay was carried out in a buffer containing phosphoenolpyruvate, NADH, pyruvate kinase, lactate dehydrogenase, and PIM kinase. The reaction was monitored at 340 nm at 25 deg C on a Spectramax spectrophotometer (Molecular Devices) and started by addition ATP after a 10-min preincubation at 25 deg C. A recognition peptide of the PIM1 substrate p21 (RKRRQTSMTD) was used. DMSO-dissolved inhibitors were added at the preincubation period resulting in a 2% final DMSO. Kinetic analysis was done by nonlinear regression fitting using the program KaleidaGraph (Synergy Software). |
| Affinity data for this assay | |
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