| Assay Method Information | |
| | HTRF Assay |
| Description: | JAK3 Kinase activity was monitored using a HTRF KinEASE-TK kit from Cisbio (62TK0PEC). The buffer used was 1 kinase buffer supplemented with 2 mM MnCl2, 5 mM MgCl2, 1 mM TCEP and 100 diluted Supplementary Enzyme Buffer. JAK-3 was purchased from Promega. 0.0133 ng/ul JAK3 (0.21 nM) was preincubated in the absence or presence of inhibitor at room temperature for 3 hours. Reaction with substrate was then initiated by adding biotinylated substrate and ATP. The concentration of substrate in the reaction mixture was 0.5 uM and ATP was 1.434 uM (reported Km value). The reaction was terminated by adding 31.25 nM SA-XL665 and 100 fold diluted europium labelled antibody (Eu-Ab), diluted in 1 detection buffer that contained EDTA. After 60 min of incubation, the fluorescence emission was measured at 620 nm and 665 nm. The ratio of Em 665 nm to Em 620 nm was proportional to the amount of substrate phosphorylated by the kinase. |
| Affinity data for this assay | |
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