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| | LANCE Ultra Assay |
| Description: | The LANCE Ultra assay kit was purchased from PerkinElmer. The ULight-MBP peptide, Europium labeled Antibody and LANCE Detection buffer were purchased from PerkinElmer. The APT and dimethylsulfoxide were purchased from Sigma-Aldrich. The 5× Kinase buffer were purchased from Invitrogen. The MAP kinase 1 (Mek1), inactive Erk1 were purchased from Millipore. A Mek1 kinase assay (LANCE, PerkinElmer) was developed for supporting compound profiling and lead optimization. In this assay, un-phosphorylated/inactive Erk1 (Millipore) was used as the substrate for Mek1 (Millipore). Then the phosphorylated Erk1 was able to phosphorylate ULight-MBP peptide (PerkinElmer). The phosphorylated peptide was detected by Europium-anti-phospho-MBP (PerkinElmer). In a reaction, the activity of Mek1 (0.5 nM) was measured in a buffer containing 50 μM ATP, 2 nM inactive Erk1, 2 nM ULight-MBP peptide, and a compound for 90 min at 23° C. After quenching the reaction with xxx, 2 nM Europium-anti-phospho-MBP was added to the reaction mixture and incubated for 60 min, followed by a detection using EnVision Multilabel Plate Reader (PerkinElmer). |
| Affinity data for this assay | |
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