| Assay Method Information | |
| | JAK2 Inhibition Assay |
| Description: | Recombinant human JAK2 (catalytic domain, amino acids 866-1154; catalog number PV4210) was purchased from Invitrogen. 0.0125 mU of JAK2 was incubated with 25 nM Ulight-JAK1(tyr1023) peptide (Perkin Elmer catalog number TRF0121) in kinase reaction buffer (41.66 mM HEPES pH7.0, 0.016% Triton X-100, 12.5 mM MgCl2, 3.33 mM DTT, 7.5M ATP) with or without 4L containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 20L, in a white 384 Luminotrac 200 plate (Greiner, catalog number 781075). After 60 min at room temperature, reactions were stopped by adding 20L/well of detection mixture (1x detection buffer (Perkin Elmer, catalog number CR97-100C), 0.5 nM Europium-anti-phosphotyrosine (PT66) (Perkin Elmer, catalog number AD0068), 10 mM EDTA). Readout is performed using the Envision with excitation at 320 nm and measuring emission at 615 nm (Perkin Elmer). Kinase activity was calculated by subtracting relative fluorescence units (RFU) obtained in the presence of a positive control inhibitor (10M staurosporine) from RFU obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as: Percentage inhibition=((RFU determined for sample with test compound present-RFU determined for sample with positive control inhibitor) divided by (RFU determined in the presence of vehicle-RFU determined for sample with positive control inhibitor))*100. Dose dilution series are prepared for compound enabling the testing of dose-response effects in the JAK2 assay and the calculation of the IC50 for the compound. Each compound is routinely tested at concentration of 20M followed by a 1/5 serial dilution, 8 points (20M-4M-800 nM-160 nM-32 nM-6.4 nM-1.28 nM-0.26 nM) in a final concentration of 1% DMSO. When potency of compound series increases, more dilutions are prepared and/or the top concentration are lowered (e.g. 5M, 1M). The data are expressed as the average IC50 from the assays standard error of the mean. |
| Affinity data for this assay | |
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