| Assay Method Information | |
| | SIK2 Enzymatic Assay High Version 2 (High ATP) |
| Description: | All steps were performed at room temperature. Compounds were pre-plated onto an assay plate using an Echo acoustic dispenser (Labcyte, E550 or E555) at 80 nL per well. Enzyme prepared in assay buffer at 6 nM (2× final concentration) was added at 4 μL per well using the Multidrop Combi (Thermo Fisher-used for all additions in this assay) to the assay plate, spun for 1 min at 1000 rpm (Allegra 6KR Beckman Coulter centrifuge), and incubated with compound for 30 minutes. Assay buffer containing 6 mM ATP and 400 μM HDAC5tide (2× of final concentration for each substrate) was added at 4 μL per well to the assay plate and then spun as above and incubated for 2 hours. Note that typically one column of wells was dedicated to a no-enzyme negative control (containing ATP and peptide) and another dedicated to DMSO only (maximal enzyme activity). ADP-Glo Max Reagent (prepared and aliquoted as per manufacturer instructions) was then added at 8 μL per well, spun and incubated for 2 hours. ADP-Glo Max Detection (prepared and aliquoted as per manufacturer instructions) was added at 8 μL per well to the assay plate, which was spun and incubated for 1 hour. The plate was then read on the Pherastar (BMG Labtech) using a luminescence read. Gain was set between 2200-2500. |
| Affinity data for this assay | |
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