| Assay Method Information | |
| | Syk Enzyme Assay |
| Description: | Syk was pre-activated at room temperature for 30 mins in the presence of 16.6 mM MgCl2, 8.3 mM ATP and then diluted to 4 nM in 40 mM Hepes pH 7.4, 0.01% BSA. 3 μl of substrate reagent containing biotinylated peptide, Biotin-AAAEEIYGEI (0.5 μM final), ATP (30 μM final) and MgCl2 (10 mM final) in 40 mM HEPES pH 7.4, 0.01% BSA, were added to wells containing 0.1 μl of various concentrations of compound or DMSO vehicle (1.7% final) in Greiner low volume 384 well black plate. The reaction was initiated by the addition of 3 μl of diluted Syk (2 nM final). The reaction was incubated for 60 min at room temperature, then terminated by the addition of 3 μl of read reagent containing 60 mM EDTA, 150 mM NaCl, 50 nM Streptavidin APC (Prozyme, San Leandro, Calif., USA), 0.5 nM antiphosphotyrosine antibody labelled with W-1024 europium chelate (Wallac OY, Turku, Finland) in 40 mM HEPES pH 7.4, 0.03% BSA. The reaction was further incubated for 45 min at room temperature. The degree of phosphorylation of Biotin-AAAEEIYGEI was measured using a BMG Rubystar plate reader (BMG LabTechnologies Ltd, Aylesbury, UK) as a ratio of specific 665 nm energy transfer signal to reference europium 620 nm signal. |
| Affinity data for this assay | |
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