| Assay Method Information | |
| | Inhibition of CLK, CLK2, CLK3 and CLK4 Kinase Activity Evaluated at Nanosyn |
| Description: | Table 3: Kinase protein and substrate were pre-diluted in the HEPES assay buffer (100 mM HEPES, pH 7.5, 0.01% Triton X-100, 0.1% BSA, 5 mM MgCl2, 1 mi DTT, 10 Sodium Orthovanadate, 10 M Beta-Glycerophosphate) dispensed into 384 well plate (5 μL per well). Control samples (0%-inhibition in the absence of inhibitor, DMSO only) and 100%-inhibition (in the absence of enzyme) were assembled in replicates of six and were used to calculate %-inhibition in the presence of compounds. Test compounds were added to the protein samples by acoustic dispensing (Labcyte Echo550). Concentration of DMSO was equalized to 1% in all samples. Reactions were initiated by addition of ATP by acoustic dispensing (Labcyte Echo550) and incubated according to assay specific incubation time. After incubation. 5 μL of Promega ADP-Glo reagent was added and incubated for 40 minutes. After 40 minutes, 10 uL of Promega kinase detection reagent was added. After 10 min of incubation with Kinase detection reagent, the luminescence was read on microplate reader (Biotek Synergy). |
| Affinity data for this assay | |
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