| Assay Method Information | |
| | End Point Fluorescence Assay |
| Description: | Reaction volumes of 50 μL were used in 96-well plates. Buffer A (1×, 34 μL; 100 mM Tris, pH 8, 10 mM MgCl2) was added to a single row, followed by 15 μL ofenzyme (3.3× concentration) in buffer A with 3-fold dilutions (typically, 125 nM and 42, 14, 4.6, 1.5, 0.5, 0.17, 0.06, 0.02, 0.01, and 0 μM final well concentrations in buffer A). Then, 1 μL of a 500 nM stock of the appropriate dasatinib analogue BODIPY probe in DMSO was added (2% DMSO final). Wells were incubated at rt for 30 min prior to end point read (ex/em 485/535 nm). Reactions had final concentrations of 10 nM BODIPY-probe, 100 mM Tris buffer (pH 8),and 10 mM MgCl2. |
| Affinity data for this assay | |
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