| Assay Method Information | |
| | Inhibition Test of the Compounds of the Invention on the JAK Kinase |
| Description: | JAK1 LANCE Ultra assay, JAK2 LANCE Ultra assay and JAK3 LANCE Ultra assay were used to carry out the test separately, and kits of the above assays were supplied by PerkinElmer.A reaction system of JAK1 LANCE Ultra kinase assay comprises 2 nM JAK1 (Intech, PV4775), 50 nM ULight™-JAK-1 peptide (substrate, PerkinElmer, TRF0121-M) and 38 uM ATP (Sigma, A7699). A reaction system of JAK2 LANCE Ultra kinase assay comprises 0.03 nM JAK2 (Intech, PV4288), 50 nM ULight™-JAK-1 peptide (substrate, PerkinElmer, TRF0121-M) and 12 uM ATP (Sigma, A7699). A reaction system of JAK3 LANCE Ultra kinase assay comprises 0.08 nM JAK3 (Intech, PV4080), 50 nM ULight™-JAK-1 peptide (substrate, PerkinElmer, TRF0121-M) and 4 uM ATP (Sigma, A7699).Buffers of an enzymatic reaction is 50 mM 4-hydroxyethylpiperazine ethane sulfonic acid (pH7.5) Intech, 15630130), 10 mM magnesium chloride (Sigma, 63020), 1 mM edetic acid (Intech, 1842C505), 2 mM dithiothreitol (Intech, 43815) and 0.01% BRIJ-35 (Intech, B4184). JAK1, JAK2 and JAK3 kinase, ATP and substrates were dissolved and diluted with buffers.A test liquid was 2 nM Eu-W1024 anti-phosphotyrosine (PerkinElmer, AD0069). A stop buffer was 10 mM edetic acid (Intech, 1842C505). |
| Affinity data for this assay | |
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