| Assay Method Information | |
| | Kinase Inhibition Activity Assay |
| Description: | LANCE (Lanthanide Chelate Excite) Eu time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay (PerkinElmer) was performed in 384-well OptiPlates (Corning) using recombinant RSK2 kinase (CarnaBio), ULight -phospho-40S ribosomal protein S6 (Ser235/236) peptide substrate (PerkinElmer), and ATP (Sigma) according to the supplier protocols. All reagents were prepared in kinase buffer containing 2 mM DTT, 50 mM HEPES, 1 mM EGTA, 10 mM MgCl2, 0.01% Tween 20, pH 7.5. Inhibitor solutions were prepared such that the final DMSO concentration did not exceed 0.5%, which was shown to have no impact on kinase activity. RSK2 was used at a final concentration of 500 μM. ULight -rpS6 substrate was used at a final concentration of 250 nM and ATP was administered at a final concentration of 3 μM. Assays were performed at 25 C. in a reaction mixture consisting of 2 μL serially diluted inhibitor solution, 4 μL kinase, 2 μL substrate, and 2 μL ATP. Reagents were incubated at room temperature for 1 h before the reaction was stopped through the addition of 5 μL of EDTA at a final concentration of 10 mM. After a 5 min incubation period, 5 μL of Eu anti-phospho-40S Ribosomal Protein S6 (Ser235/236) antibody (PerkinElmer) at a final concentration of 2 nM was added. The plate was read using a Biotek Synergy H1 Hybrid plate reader (Excitation=340 nm; Substrate emission=665 nm; Antibody emission=615 nm; Delay=100 μs; Integration=200 μs). Emission ratios (665 nm/615 nm) were calculated for each well and half-maximal inhibitory concentrations (IC50) were determined for each inhibitor through non-linear regression analysis of the log dose-response curves. |
| Affinity data for this assay | |
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