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| | Time Resolved-FRET Assay |
| Description: | Activity of compounds was routinely confirmed using a secondary assay as described herein. The secondary assay was a time resolved-FRET kinase assay. Test compound are diluted to desired concentrations in kinase reaction buffer and briefly incubated with BTK kinase (as described above; Invitrogen). The reaction is initiated by the addition of ATP and enzyme substrate, HTRF® KinEASE-TK Substrate-biotin (Cisbio US, Bedford, Mass.). The composition of the reaction (10 μl) was: 1% v/v DMSO, 10 nM BTK, 60 μM ATP, and 1 μM substrate. After incubation at room temperature for 60 min, the enzyme reaction is stopped by EDTA-containing buffer, which also contains europium-labeled (Eu3+-Cryptate) anti-phosphotyrosine antibody (Cisbio) and Streptavidin-XL665 (Cisbio). The europium-labeled antibody generates a time-resolved FRET signal with the Stepavidin-XL665, which binds to the biotinylated TK substrate through the streptavidin conjugate when the substrate is phosphorylated. |
| Affinity data for this assay | |
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