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Assay Method Information |
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| | Competition Binding Assays |
| Description: | Briefly, 5 mg of a protein mixture of the four cell lines or a single cell line were incubated with compound dilution series in DMSO (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 30 μM, and DMSO) or single compound dose (5 μM) for 45 min at 4 °C. This preincubation step was followed by incubation with kinobeads (35 μL settled beads). Bound proteins were eluted with LDS sample buffer (NuPAGE,Invitrogen) containing 50 mM DTT. For the calculation of a correction factor, the flowthrough of the DMSO control was incubated with fresh beads for a second time (pulldown of pulldown). |
| Affinity data for this assay | |
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