| Assay Method Information | |
| | Enzymatic Activity Assay |
| Description: | The compounds were tested for SGK-1 activity by measuring the ability of the compound to inhibit the transfer of phosphate from ATP by the isolated enzyme to serine/threonine residues in a fluorescein labeled substrate peptide, FLPeptide 6 (PerkinElmer, Waltham, USA, Cat. No: 760350). The enzymatic reaction was initiated by addition of 15 μL of solution 1 containing (in mM) 10 MgCl2, 0.010% Brij-35, 2 DTT, 0.05% BSA, 1 EGTA, 50 HEPE (pH7.5) and 0.665 nM SGK to 5 μL of solution 2 containing 10 MgCl2, 0.010% of Brij-35, 2 DTT, 0.05% BSA, 1 EGTA, 50 HEPES (pH7.5), 6 UM of FLPeptide and 80 UM of ATP. After incubating the plate at room temperature for 90 min, 75 μL of stopping buffer (containing 0.5 M EDTA) is added to terminate the reaction. The samples were analyzed using an EZ reader. For the determination of the compound dose response, stock solution of compound prepared in DMSO was diluted and tested in a 10 point, three-fold dilution series run in duplicate beginning at 10 μM final concentration. |
| Affinity data for this assay | |
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