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Assay Method Information |
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| | Inhibition Assay |
| Description: | Compounds of the invention were initially diluted to 10 mM in 100% DMSO (CALBIOCHEM) for storage and made into kinase buffer solution to create a compound concentration ranging from 1 uM and 10 uM. Serial dilutions of compounds of the invention were dispensed into a 96-well plate (GREINER BIOSCIENCES) at 6 uL each. Purified full-length human SYK (CARNA BIOSCIENCES) were diluted in kinase buffer and added to the compound solutions and pre-incubated for 30 minutes at room temperature. Next, ATP (TEKNOVA) of Km (15 uM) and substrate solution (suggested manufacture substrates of PerkinElmer, Ulight-TK peptide for SYK) was added (12 uL each) to the wells containing the compound solution and enzyme. The reaction mixture was incubated for 1 hour. Following the incubation, the stop solution made with EDTA, water, and Lance detection buffer (PERKINELMER) was added (12 uL each) to stop phosphorylation. Following the addition of the stop solution and 5 minutes of shaking. |
| Affinity data for this assay | |
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