| Assay Method Information | |
| | Isothermal Titration Calorimetry (ITC) |
| Description: | CDK2 was thawed at room temperature and buffer exchanged using Zeba spin desalting columns. The final ITC buffer was 1×PBS, 10 mM MgCl2, and 5% glycerol (pH=7.4), with 5% DMSO added after buffer exchange into the protein solution and from a 1:20 concentrated DMSO stock for the ligand solution. CDK2 concentration was determined using a NanoDrop™ spectrophotometer and calculated using absorption at 280 nm (ε=36,900 M−1 cm−1). Into a 96-well plate for automatic injection for the MicroCal iTC200 (Malvern), a final volume of 400 μL protein solution for the sample cell and 200 μL inhibitor solution for the injecting syringe were placed into the appropriate wells. The final inhibitor concentration in the syringe was 500-1,000 μM depending on compound solubility. For Compounds 3-7, to get more accurate data on the ITC with appropriate c values (Wiseman, T., et al., Anal. Biochem. 1989, 179 (1), 131-7), the CDK2 concentration was lowered about 10-fold to 5 μM and the syringe concentration lowered to 75 μM. The ITC experiments were conducted at 25° C. and 750 rpm stirring, with a discarded initial 0.4 μL injection and 20 subsequent 4 μL injections with 180 s between injections. A one-site binding model was used to fit the ITC data after adjusting the baseline to account for slight buffer mismatch between the cell and syringe samples. All ITC runs were done in duplicates (n=2) for each compound. |
| Affinity data for this assay | |
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