| Assay Method Information | |
| | In Vitro CDK9/CyclinE1 Enzyme Activity Assay |
| Description: | Experimental Methods:Preparation of kinase buffer: the kinase buffer containing 50 mM HEPES, 1 mM EDTA, 10 mM MgCl2, 0.01% Brij-35, pH 7.4.2.38 g of HEPES, 58 mg of EDTA, 406 mg of MgCl2, and 20 mg of Brij-35 were added to 200 mL of buffer, and the pH was adjusted to 7.4.Preparation of Stop Solution:100 μL of 1 M EDTA stock solution was mixed with 0.625 μL of 1× detection buffer and 1725 μL of distilled water to prepare the stop solution.Enzyme, Ulight-4E-BP1 peptide, ATP, and inhibitors were diluted with the kinase buffer.The Eu-anti-phospho-tyrosine antibody was diluted to a concentration of 8 nM/L using detection buffer.The compounds to be tested were 4-fold diluted with a multi-channel pipette to the 8th concentration, i.e., from 400 μM to 24.4 nM, with a final DMSO concentration of 4%. The experiment was set up in duplicate wells. 2.5 μL of each concentration gradient of the inhibitor was added to the microplate, followed by 5 μL of CDK9-CyclinT1 enzyme (2 ng), and 2.5 μL of a mixture of substrate and ATP (8 mM ATP, 50 nM Ulight-4E-BP1 peptide). At this point, the final concentration gradient of the compound was 100 μM diluted to 6.1 nM. The reaction system was reacted at 25° C. for 120 minutes. After the reaction, 5 μL of stop solution was added to each well, and the reaction was continued at 25° C. for 5 minutes. After stopping the reaction, 5 μL of Eu-anti-phospho-tyrosine antibody dilution was added to each well, and the mixture was reacted at 25° C. for 60 minutes. Data were then collected using the PerkinElmer Nivo multimode microplate reader in TR-FRET mode (excitation wavelength: 320 nm, emission wavelength: 665 nm).Experimental Methods:Preparation of kinase buffer: the kinase buffer containing 50 mM HEPES, 1 mM EDTA, 10 mM MgCl2, 0.01% Brij-35, pH 7.4.2.38 g of HEPES, 58 mg of EDTA, 406 mg of MgCl2, and 20 mg of Brij-35 were added to 200 mL of buffer, and the pH was adjusted to 7.4.Preparation of Stop Solution:100 μL of 1 M EDTA stock solution was mixed with 0.625 μL of 1× detection buffer and 1725 μL of distilled water to prepare the stop solution.Enzyme, Ulight-4E-BP1 peptide, ATP, and inhibitors were diluted with the kinase buffer.The Eu-anti-phospho-tyrosine antibody was diluted to a concentration of 8 nM/L using detection buffer.The compounds to be tested were 4-fold diluted with a multi-channel pipette to the 8th concentration, i.e., from 400 μM to 24.4 nM, with a final DMSO concentration of 4%. The experiment was set up in duplicate wells. 2.5 μL of each concentration gradient of the inhibitor was added to the microplate, followed by 5 μL of CDK9-CyclinT1 enzyme (2 ng), and 2.5 μL of a mixture of substrate and ATP (8 mM ATP, 50 nM Ulight-4E-BP1 peptide). At this point, the final concentration gradient of the compound was 100 μM diluted to 6.1 nM. The reaction system was reacted at 25° C. for 120 minutes. After the reaction, 5 μL of stop solution was added to each well, and the reaction was continued at 25° C. for 5 minutes. After stopping the reaction, 5 μL of Eu-anti-phospho-tyrosine antibody dilution was added to each well, and the mixture was reacted at 25° C. for 60 minutes. Data were then collected using the PerkinElmer Nivo multimode microplate reader in TR-FRET mode (excitation wavelength: 320 nm, emission wavelength: 665 nm). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |