| Assay Method Information | |
| | Kinetic Assay with Fluorescence Anisotropy |
| Description: | Association kinetics (Kon): All measurements were performed in HEPES (50 mM, pH 7.5) containing NaCl (150 mM), Tween-20 (0.005%) and DTT (5 mM) according to a previously published protocol.[Vaasa et al., Anal. Biochem., 385:85-93] The final volume of the assay mixture was 20 mL.All measurements were performed in HEPES(50 mM, pH 7.5) containing NaCl (150 mM), Tween-20 (0.005%) and DTT (5 mM). The association kinetics assay with fluorescent probes and data analysis were performed according to published protocols.[Vaasa et al., Anal. Biochem., 385:85-93] The final volume of the assay mixture was 20 mL. Dissociation kinetics (Koff): All measurements were performed in HEPES (50 mM, pH 7.5) containing NaCl (150 mM), Tween-20 0.005%) and DTT (5 mM). Data analysis was as reported previously,[Vaasa et al., Anal. Biochem., 385:85-93] by using method1 for compounds with shorter residence times, and method 2 for 1 in studies with haspin. The final volume of the assay mixture was 20 mL. Kd' is calculated as Koff/Kon. |
| Affinity data for this assay | |
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