| Assay Method Information | |
| | ADP-GLO Kinase Assay |
| Description: | Briefly, 4 compound solution and 4 ATP solution are prepared with assay buffer (50 mM MOPS, 1 mM DTT, pH7.2). 4 Substrate solution and 4 kinase/Metal solution are prepared with MOPS based buffer containing individual kinase specific additives. Then, L of 4 compound solution, 5 μL of 4 Substrate solution, 5 μL of 4 ATP solution, and 5 μL of 4 kinase/Metal solution are mixed and incubated in a well of polystyrene 384 well black microplate for 1 hour at room temperature. Next, 20 μL of ADP-Glo Reagent (Promega) is added to the well,and incubated for over 40 minutes. 40 μL of Kinase Detection Reagent (Promega) is added to the well, and incubated for over 40 minutes. The kinase reaction is evaluated by the endpoint luminescence of the well. For measurement of PIKFYVE activity, the substrate is PI3P at 10,000 nM concentration, using AG-182 as a positive control. The metal used is magnesium at 5 mM concentration. |
| Affinity data for this assay | |
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