| Assay Method Information | |
| | In Vitro CDK7 Assay |
| Description: | CDK7 activity is measured by following the production of ADP generated from ATP-dependent phosphorylation of the peptide substrate derived from RNA Pol II (CDK7/9 tide) by CDK7. Pyruvate kinase converts ADP and phosphoenolpyruvate (PEP) to ATP and pyruvate. Lactase dehydrogenase catalyzes pyruvate to lactate with a concomitant conversion of NADH to oxidized form NAD+, which is spectrophotometrically measured at 340 nm. The CDK7 assay was performed in 384-well microplates with a final volume of 100 μL. Inhibitor serial dilutions and liquid handing for the assay were performed by using Janus from PerkinElmer (Downers Grove, IL) and Tempest from Formulatrix (Bedford, MA), respectively. To determine inhibitor potency of irreversible covalent inhibitors (kinact/K1 ratios), 500 nL of inhibitor in DMSO (or DMSO for controls) was added to the assay plate using Echo 555 from Labcyte (San Jose, CA) followed by 50 μL of assay mixture consisting of 600 μM peptide substrate (CDK7/9 tide, YSPTSPSYSPTSPSYSPTSPSKKKK), 1 mM ATP, 1 mM PEP, 200 μM NADH, 1.2-2 units of PK, 1.8-2.8 units of LDH, 20 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 0.004% Triton X-100. Reactions were initiated by the addition of 50 μL of 40 nM CDK7/cyclinH/MAT1 trimeric complex in 20 mM Tri-HCl (pH 7.4), 10 mM MgCl2, and 0.004% Triton X-100. The assay plates were centrifuged at 3220 g for 5 min using Centrifuge 5810 from Eppendorf (Hauppauge, NY) and then the absorbance changes were read at 340 nm at room temperature using Infinite M1000 from Tecan (Mannedorf, Switzerland) every 2 min for 8 hours. |
| Affinity data for this assay | |
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