| Assay Method Information | |
| | Kinase Assay |
| Description: | Btk (human, full length) was purchased (Invitrogen, catalog number PV3363) and used as specified in the product literature. Btk (2 nM final concentration) was pre-incubated with inhibitors (six or ten concentrations, in duplicate) for 30 minutes at room temperature. Kinase reactions were initiated by the addition of 0.16 μCi/μL of γ-32P-ATP (6000 Ci/mmol, NEN) and 0.2 mg/mL substrate (poly[Glu:Tyr], 4:1 Glu:Tyr) and incubated for 30 minutes at room temperature. Kinase activity was determined by spotting 6 μL of each reaction onto sheets of phosphocellulose. Each blot was washed once with 1% AcOH solution, twice with 0.1% H3PO4 solution, and once with MeOH (5-10 minutes per wash). Dried blots were exposed for 30 minutes to a storage phosphor screen and scanned by a Typhoon imager (GE Life Sciences). |
| Affinity data for this assay | |
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