| Assay Method Information | |
| | Wee1 Enzymatic Activity Inhibition Assay |
| Description: | The enzymatic assay used to determine activity was a Luminescence assay using a Microplate Reader (BMG, ClarioStar Plus). The enzymatic reaction was carried out in assay buffer (40 mM TRIS-HCl pH 7.4-7.6, 20 mM MgCl2, 0.05 mM DTT, 0.1 mg/ml BSA, 5 mM MnCl2). The compounds were dispensed on a 384 well Diamond Well Plate (Axigen, Cat #P-384-120SQ-C-S) using the Biomek FX liquid handling system at 100× solutions of compounds in DMSO. 2×Wee1-PolyE4Y1 mix (final concentration 0.85 ng/μl of Wee1 and 0.2 μg/μl of PolyE4Y1) was prepared in 1× Assay buffer and 5.5 μl of mixture per well was added into 384w white Reaction plate with NBS (Corning, Cat #4513). 5.5 μl of PolyE4Y1 substrate w/o Wee1 in 1× buffer was used for negative control. Plates were centrifuged for 1 min at 100 g. Next step the Compounds were added to Reaction plate using Biomek station via following steps: 1 μl of 100× compounds (in DMSO) were mixed thoroughly with 49 μl of 2×10 μM ATP in Assay Buffer, then 5.5 μl of this mixture was added to Reaction plate with 5.5 μl of Wee1-PolyE4Y1 mix. Plates were centrifuged for 1 min at 100 g and incubated for 1 h at room temperature. Next 3 μL of ADP-Glo reagent (Promega, ADP-Glo™ Kinase Assay, Cat #V9102) per well was added. Plates were incubated for 30 minutes at room temperature. Then 6 μL of Kinase detection reagent (Promega, ADP-Glo™ Kinase Assay, Cat #V9102) per well was added and the Luminescence was measured using Microplate Reader. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |