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| | TR-FRET Competition Assay |
| Description: | Assay buffer was 20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.01% BSA, 1 mM DTT, 0.0005% Tween 20, and 2% DMSO. Inactivation kinetic reactions were performed by preparing 15 μL of a 1.33Χ solution of (final concentrations) 2 nM Eu-Ab, 1-8 nM kinase (optimal concentrations of each kinase were empirically determined) and a variable concentration of PF- 06651600, and pre-incubating this for a variable amount of time (detailed below). This was then combined this with 5 μL of 4X solution of the validated probe (150 nM, final concentration). For all kinases, the following experiments were performed: (A) [PF-06651500] = 0, 4.9, 14.8, 44.4, 133.3, and 400 nM; pre-incubation time = 2 h. (B) [PF-06651500] = 0, 0.5, 1.0, 2.0, 4.0, and 8.0 μM; pre-incubation time = 120 s. For JAK3, the following additional experiments were performed: [PF-06651500] = 0, 0.66, 1.98, 5.93, 17.8, 53.3, 160, 480 nM; pre-incubation time of (C) 30 s, (D) 60 s, and (E) 1.5 h. The assays were read using an EnVi |
| Affinity data for this assay | |
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