| Assay Method Information | |
| | CDK1, CDK2, and CDK4 Activity Assay |
| Description: | Compounds were tested in breast cancer cell line MCF-7 to assess inhibition of CDK1, CDK2, and CDK4 activity. MCF-7 cells transiently transfected with CDK1:CCNB1, CDK2:CCNE1, or CDK4:CCND1 were harvested at a density of 1E5 cells/mL in complete media, seeded 20 μL/well into 384-well Greiner 784080 plates using a Multidrop Combi, and incubated overnight at 37° C. and 5% CO2. The next day media were evacuated from the wells using the Bluewasher centrifugal plate washer (Bluecatbio) and 10 μL/well phenol red free OptiMEM was then added using a Multidrop Combi. Test compounds were then dispensed into the wells using an Echo instrument (555/655, Beckman Coulter). Immediately after compound addition, a Tecan HP300 dispensor was used to dispense the relevant NanoBRET™ tracer to the CDK1 wells (12.5 nl, 400 μM, NanoBRET™ TE Tracer K-9), the CDK2 wells (12.5 nl, 200 μM, NanoBRET™ TE Tracer K-9), and the CDK4 wells (8 nl, 100 μM, NanoBRET™ TE Tracer K-7) and the plates were incubated for 2 hours at 37° C. and 5% CO2. The plates were allowed to cool for 10 minutes at room temperature and 5 μL/well of TE Nano-Glo®Substrate/Inhibitor at 2.4 μM and 1:500 respectively (N2162 Promega) was added. The plates were incubated in subdued lighting for 10 minutes and then read on a Pherastar FS plate reader (BMG Technologies) using a NanoBRET™ filter module (460±80 nm/610 nm-LP). The ratio values were normalized to controls and the IC50 values of test compounds determined using Genedata Screener software. |
| Affinity data for this assay | |
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