| Assay Method Information | |
| | Biochemical Assay |
| Description: | To perform the enzymatic step, working solutions were prepared in enzymatic buffer (12.5 nM SEB, 1 mM DTT, 5 mM MgCl2, 1 mM MnCl2). The reaction was carried out in a 384-well plate with 2.5 μL inhibitor or enzymatic buffer, 2.5 μL recombinant human protein (CSF-1R, c-Kit, PDGFRβ and FLT3) and 5 μL substrate/ATP mixture at room temperature for 1 hour. After addition of detection mixture (5 μl Sa-XL 665 and 5 μl TK-antibody-Cryptate), the plate is sealed and incubated at room temperature for 1 hour. Detection was performed with Spark reader (Tecan) using standard HTRF protocol settings. IC50 determinations were performed using GraphPad Prism software (GraphPad, Inc.). The selectivity fold was determined by the IC50 of other kinases (cKit, FLT3, PDGFR-1β) and a multiple of the IC50 of CSF-1R. |
| Affinity data for this assay | |
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