| Assay Method Information | |
| | Isothermal Titration Calorimetry (ITC) |
| Description: | Calorimetric titrations were performed in a Microcal iTC 200 microcalorimeter. Freshly purified 5 µM protein (Aurora A/B kinases) was titrated against 100 µM ligand (felodipine/ PTK66) in each case at 25 °C. Proteins and the ligandswere diluted in the final dialysate (20 mM Tris-HCl, pH 8.0, 0.2 mM EDTA and 100 mM KCl) against which the proteins were extensively dialysed to avoid any artefact due to the minor differences in buffer composition. DMSO content was kept exactly same in the cell (not exceeding 0.2% v/v) and syringe to rule out any interference due to DMSO content mismatch. All samples were thoroughly degassed prior to titration and were prepared with the same batch of dialysate to avoid any artefacts from the minor differences in buffer composition. Dilutions of 100 µM felodipine/PTK66 in dialysate containing equal percentage of DMSOserved as controls. The stirring speed was 300 rpm. |
| Affinity data for this assay | |
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