| Assay Method Information | |
| | Biological Assay |
| Description: | Compounds of the present disclosure were tested in a 10-dose IC50 mode with a 3-fold serial dilution starting from 0.5 μM, and the control compound Staurosporine was tested in a 10-dose IC50 mode with a 4-fold serial dilution starting from 20 μM. Reactions were carried out in the presence of 10 μM ATP. Conditions and Protocol: Procedures:1. Compounds were prepared in freshly prepared reaction buffers containing 20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.01% Brij35, 0.02 mg/ml, BSA, 0.1 mM Na3VO4, 2 mM DTT, and 1% DMSO.2. Required cofactor such as 1 μg (1.5 μM) of recombinant retinoblastoma protein in the case of CDK subtypes was added to the substrate solutions as mentioned above.3. Kinase such as 10 ng of recombinant CDK4/cyclin D1 (Life Technologies PV4204) diluted in a kinase buffer (20 mM Tris pH7.5, 10 mM MgCl2, 0.01% NP-40, 2 mM DTT), and incubated at room temperature for 30 minutes together with indicated concentration of inhibitors.4. Compounds in DMSO were added into the kinase reaction mixture utilizing acoustic technology (Echo550).5. 33P-ATP (specific activity 0.01 μCi/μl final) was added into the reaction mixture to initiate the reaction.6. The reaction mixture was incubated for 120 minutes at room temperature.7. Reactions are spotted onto P81 ion exchange paper (Whatman #3698-915).8. Filters were extensively washed with 0.75% phosphoric acid.9. The radioactive phosphorylated substrate remaining on the filter paper was measured. |
| Affinity data for this assay | |
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