| Assay Method Information | |
| | JAK2 Enzymatic Assay |
| Description: | The assay has been performed using the commercial available JAK2 kinase domain (Invitrogen, Eugene, Oreg.). The JAK2 kinase domain showed a linear kinetic without prephosphorylation. The JAK2 kinase assay was run with a final enzyme concentration of 1 nM, in the presence of 60 uM ATP, 3 nM 33P-γ-ATP and 64 uM of substrate BioDBn*306 (Aminoacid sequence: LPLDKDYYWREPGQ SEQ ID NO: 1). The peptidic substrate was purchased from American Peptide Company (Sunnyvale, Calif.). Specific JAK2, JAK1 or JAK3 peptide substrates are trans-phosphorylated by JAKs kinase in the presence of ATP traced with 33P-γ-ATP. At the end of the phosphorylation reaction, the unreacted ATP, cold and radioactive, is captured by an excess of Dowex ion exchange resin that eventually settles by gravity to the bottom of the reaction plate. The supernatant is subsequently withdrawn and transferred into a counting plate that is then evaluated by 3-counting. |
| Affinity data for this assay | |
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