| Assay Method Information | |
| | JAK1 Inhibition Assay |
| Description: | Recombinant human JAK1 (catalytic domain, amino acids 866-1154; catalog number PV4774) was purchased from Invitrogen. 1 ng of JAK1 was incubated with 20 nM Ulight-JAK1(tyr1023) peptide (Perkin Elmer catalog number TRF0121) in kinase reaction buffer (25 mM MOPS pH6.8, 0.016% Brij-35, 8.33 mM MgCl2, 3.33 mM DTT, 7 uM ATP) with or without 4 uL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 20 uL, in a white 384 Luminotrac 200 plate (Greiner, catalog number 781075). After 60 min at room temperature, reactions were stopped by adding 20 uL/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 (10 uM 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 were prepared for the compounds enabling the testing of dose-response effects in the JAK1 assay and the calculation of the IC50 for the compound. Each compound is routinely tested at concentration of 20 uM followed by a 1/5 serial dilution, 8 points (20 uM-4 uM-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. 5 uM, 1 uM). 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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