| 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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