| Assay Method Information | |
| | HASPIN biochemical Assay |
| Description: | The biochemical assay to measure HASPIN activity relies on the ADP-Glo assay kit (Promega) that determines the amount of ADP as direct product of the kinase enzyme activity. Assay conditions were as indicated by the kit manufacturers with the following adaptations for the kinase activity step: Kinase assay buffer and assay volume (15 mM HEPES pH 7.5, 20 mM NaCl, 1 mM EGTA, 0.02% TWEEN 20, 10 mM MgCl2, 0.1 mg/ml BGG)/25 uL assay volume). Incubation time and temperature: 60 min at 30 C. HASPIN final concentration: 0.9 ug/mL. ATP final concentration: 150 uM. HASPIN autophosphorylation was measured. Assays were performed in 384-well plates. The final outcome of the coupled reactions provided by the kit is the release of luciferase and has been measured with a multilabel HTS counter Victor V/Envision. Values were normalized against the control activity included (100% HASPIN activity, without compound). Values were plotted against the inhibitor concentration and fit to a sigmoid dose-response curve using Activity base by IDBS software. |
| Affinity data for this assay | |
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