| Assay Method Information | |
| | In Vitro SIK2 Kinase Assay |
| Description: | Assays were performed in base reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO). Compounds were dissolved in 100% DMSO in a 10 mM stock. Serial dilution was conducted by Integra Viaflo Assist in DMSO. Recombinant SIK2 was used at a concentration of 2.5 nM. The substrate used was pAMARA at a concentration of 0.2 mg/mL. Kinase assays were supplemented with 2 mM Mn2+, and 1 μM ATP was added. Assays were performed for 20 minutes at room temperature, after which time 33P-ATP (10 μCi/μL) was added followed by incubation for another 120 minutes at room temperature. Thereafter, radioactivity incorporated into the pEY peptide substrate was detected by filter-binding method. Kinase activity data were expressed as the percent remaining kinase activity in test samples compared to DMSO reactions. IC50 values and curve fits were obtained using Prism (GraphPad Software). |
| Affinity data for this assay | |
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