| Assay Method Information | |
| | JAK2 Kinase Inhibitory Activity Assay |
| Description: | 1) Treatment wells of the compound to be tested (T-compound): In HTRF 96 well low volume plate, 4 μL of 2.5×the compound to be tested described above was added to a well, followed by the addition of 2 μL of 5×substrate to one side of the well and the addition of 2 μL of 5×JAK2 to the other side of the well.DMSO control wells without the compound to be tested (T-enzyme): In HTRF 96 well low volume plate, 4 μL of 2.5×DMSO described above was added to a well, followed by the addition of 2 μL of 5×substrate to one side of the well and the addition of 2 μL of 5×JAK2 to the other side of the well.Enzyme-free blank control (T-without enzyme): In HTRF 96 well low volume plate, 4 μL of 2.5×DMSO described above was added to a well, followed by the addition of 2 μL of 5×substrate to one side of the well and the addition of 2 μL of 1×kinase buffer to the other side of the well.2) The plate was sealed with a plate sealing film, placed into a centrifuge, and centrifuged at 1000 rpm for 2 minutes.3) 2 μL of 5×ATP was added into each well, and the plate was sealed with a plate sealing film, centrifuged at 1000 rpm for 1 minute, and then incubated in an incubator at 30° C. for 2 h.4) After the incubation, Streptavidin-XL665 and 1×TK-Antibody-Eu3-Cryptate described above were mixed at a ratio of 1:1, 10 μL of the mixture was added into each well, and then the plate was centrifuged at 1000 rpm for 1 minute.5) The plate was put back into the incubator and incubated for another 1 h. After the incubation, HTRF 620/665 signals were read on a multifunctional microplate reader. |
| Affinity data for this assay | |
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