| Assay Method Information | |
| | LanthaScreen Eu Kinase Activity Assay |
| Description: | The effects of compounds on the kinases DDR1 and DDR2 were assessed by using a LanthaScreen Eu kinase activity assay technology (Invitrogen, Carlsbad, CA, USA). Kinase reactions were performed in a 10 μL volume in low-volume 384-wellplate. Reaction buffer for the kinase reaction consists of 50 mm HEPES pH 7.5, 0.01% BRIJ-35, 10 mm MgCl2, and 1 mm EGTA. The concentration of fluorescein-polyGAT substrate (Invitrogen) in the assay was 100 mm. Kinase reactions were initiated with the addition of 100 mm ATP in the presence of serial dilutions of compounds. The reactions were allowed to proceed for 1 hour at room temperature before a 10 μL preparation of EDTA (20 mm) and Eu-labeled antibody (4 nm) in TR-FRET dilution buffer was added. The final concentration of antibody in the assay well was 2 nm, and the final concentration of EDTA was 10 mm. The plate was allowed to incubate at room temperature for one more hour before the TR-FRETemission ratios of 665 nm/340 nm were acquired on a PerkinElmer EnVision multilabel reader (Perkin Elmer, Inc., Waltham, MA, USA). |
| Affinity data for this assay | |
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