| Assay Method Information | |
| | CDK4/CyclinD1 Enzyme Reaction System |
| Description: | Preparation of Kinase Buffer: The composition of the buffer: 50 mM of hydroxyethylpiperazine ethanesulfonic acid solution at pH 7.5, 1 mM of ethylenediaminetetraacetic acid, 10 mM of magnesium chloride, 0.01% Brij-35, and 2 mM of dithiothreitol. Enzymes, substrate LANCE Ultra ULight -4E-BP-1(Thr37146) Peptide, ATP, and inhibitors were diluted by kinase buffer. The compounds to be tested were diluted 5-fold with a multi-channel pipette to the eighth concentration, that is, from 40 μM to 0.512 nM, the concentration of DMSO was 4%, and a double-hole experiment was set up. 2.5 μL of each concentration gradient of inhibitors and 5 μL of CDK4/CyclinD1 enzyme (0.5 ng) were added to the microplates, after the reaction was carried out at 25 C. for 60 minutes, 2.5 μL mixture of substrate and ATP (350 μM ATP, 12.5 nM substance) were added, and the final concentration gradient of the compound was 10 μM to 0.128 nM. The reaction system was reacted at 25 C. for 120 minutes. After the reaction, 5 μL mixture of EDTA and 2 LANCE Detection Buffer (1:1) were added to each well, the reaction was carried out at 25 C. for 5 minutes, after the reaction was completed, 5 μL of LANCE Ultra Eu-anti-P-4E-BP1(Thr37MS) (4 nM) was added to each well, the reaction was carried out at 25 C. for 60 minutes, the reaction signal was detected by Nivo instrument according to the principle of time-resolved fluorescence resonance energy transfer. |
| Affinity data for this assay | |
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