| Assay Method Information | |
| | n Vitro CDK7 (20 nM) Assay |
| Description: | Inhibition potencies of compounds were studied using an absorbance kinetic assay as described below. Compounds with potencies approaching the limit of detection of the assay (IC50 < 10 nM) were further assessed in a more sensitive fluorescence end-point assay. Absorbance kinetic assay (20 nM CDK7/Cyclin H/MAT-1 complex}CDK7 complex catalyzes the ATP-dependent phosphorylation of a peptide substrate CDK7/9-tide that is derived from RNA Pol II to produce phosphorylated peptide and ADP. The kinase reaction product ADP was converted to lactate and NAD+ in the presence of phosphoenol pyruvate (PEP), NADH and coupling enzymes lactate dehydrogenase (LDH) and pyruvate kinase (PK). CDK7 complex catalytic activity was measured by following the absorbance intensity continuously at 340 nm that corresponds with the depletion of NADH.Compound potencies were measured by a 12-point dose response manner under the assay conditions of 300 μM CDK7/9 tide (KMpeptide =140.5 ± 18.5 μM), 500μM ATP (KMATP = 27.8 ± 4.1 μM), 500 μM PEP, 100 μM NADH, 0.6-lunit PK/0.9-1.4 unit and 20 nM CDK7/Cyclin H/MAT-1 complex in a buffer containing 20 mM Tris, pH 7.4, 10 mM MgCF and 0.004% Triton X-100. Absorbance at 340 nm was followed kinetically at an interval of 2 minutes for 8 hours.The assay was carried out with 100 μl reaction volume per well in a 384-well plate that was pre-spotted nanoliter volume of compounds by LabCyte Echo 555. Compound dilution plates were made by 2-fold (could vary upon necessity) dilution in DMSO for 11 concentrations plus a DMSO control of uninhibited reaction. 2x substrate and coupling reagent mixture was added to the assay plate followed by an addition of an equal volume of 40 nM CDK7/Cyclin H/MAT-1 complex. After mixing, assay plates were spun at 2000 rpm for 3 minutes and then transferred to the plate reader for data collection. |
| Affinity data for this assay | |
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