| Assay Method Information | |
| | In Vitro Assays for Determining the Inhibition of Janus Kinases |
| Description: | The in vitro inhibition of recombinant human JAKs was determined as follow.Compounds were tested in LANTHASCREEN time-resolved fluorescence energy transfer (TR-FRET) enzymatic assays from Invitrogen. The human Janus kinase 1 (JAK1) used in the assay is the recombinant JAK1 catalytic domain (amino acids 866-1154) expressed and purified from insect cells (Invitrogen, Cat. No. PV4774). The human Janus kinase 2 (JAK2) used in the assay is the recombinant JAK2 catalytic domain (amino acids 808-1132) expressed and purified from insect cells (Invitrogen, Cat. No. PV4210). The human Janus kinase 3 (JAK3) used in the assay is the recombinant JAK3 catalytic domain (amino acids 781-1124) expressed and purified from insect cells (Invitrogen, Cat. No. PV3855). The human tyrosine kinase 2 (TYK2) used in the assay is the recombinant TYK2 catalytic domain (amino acids 833-1187) expressed and purified from insect cells (Invitrogen, Cat. No. PV4790). The substrate is a recombinant STAT1 expressed as a fusion with GFP (Green Fluorescent Protein) to act as a physiological substrate (Invitrogen, Cat. No. PV5211).Test compounds were prepared and diluted in DMSO in 3-fold serial dilutions for 10 doses to 100× of the final testing concentrations. The compounds were then further diluted to 4× by the kinase reaction buffer (Invitrogen, Cat. No. PV3189). The enzymatic reaction for compound testing was performed in a white 384-well polypropylene plate (Packard, Cat. No. 6005214) with a total reaction volume of 10 μL containing 440 ng/mL JAK1, 11 ng/mL JAK2, 400 ng/mL JAK3, or 200 ng/mL TYK2, 100 nM substrate, and 1 mM ATP. The assay started with loading 2.5 μL of JAK1, JAK2, JAK3, or TYK2 diluted in the kinase reaction buffer to wells, followed by addition of an equal volume of 4× compounds for 15-min incubation at room temperature for pre-treatment. The enzymatic reaction was initiated by addition of 5 μL of a mixture of the substrate and ATP prepared in the kinase reaction buffer. After one hour reaction, 10 μL mixture of EDTA (final 10 mM) and terbium-labeled anti-pSTAT1 (pTyr701) antibody (final 2 nM) (Invitrogen, Cat. No. PV4844) prepared in TR-FRET antibody dilution buffer (Invitrogen, Cat. No. PV3574) was added to stop the enzymatic reaction and produce TR-FRET signals. After 30 minutes of incubation at room temperature, the plate was read in Tecan Infinite F200 Pro with the following settings: Excitation 340 nm (30)/Emission1 495 nm (10)/Emission2 520 nm (25). The TR-FRET values were dimensionless numbers that were calculated as the ratio of the acceptor (Green Fluorescent Protein) signal to the donor (Terbium) signal. Percent of inhibition was calculated as (100%−percentage of compound-treated/DMSO vehicle-treated). The dose-response curves were generated and the IC50s were calculated by nonlinear sigmoid curve fitting using GraphPad Prism. |
| Affinity data for this assay | |
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