| Assay Method Information | |
| | In Vitro JAK Kinase Assay |
| Description: | The inhibition potency of the compounds was determined in JAK1, JAK2, JAK3 and TYK2 enzyme activity assays that measure peptide phosphorylation using FRET to detect product formation. Compounds were serially diluted in DMSO and 0.1 ul was transferred to the wells of black, regular volume 384-well plate by ECHO liquid handler (Labcyte, San Jose, CA) in duplicate.For all kinase assays a 10 ul volume of enzyme in assay buffer (50 mM Tris, pH=7.5, 10 mM MgCl2, 0.01% Brij-35, 0.1% BSA, 1 mM EGTA, 5% Glycerol and 5 mM DTT) was added to the plate. Reaction was started by addition of 10 ul assay buffer containing biotinylated peptide substrate (Biot-EQEDEPEGDYFEWLE-amide, Sigma-Aldrich) and ATP (final concentrations 500 nM and 1 mM respectively). Final concentrations of enzymes were as follows: JAK-1 (In-house, see ref. Blood. 2010 Apr. 15; (15) 3109)—2.73 nM, JAK-2 (BPS, Cat. #40450)—0.025 nM, JAK-3 (Carna, Cat. #08-046)—0.01 nM, TYK-2 (Invitrogen, Cat. #PR8440C)—1.75 nM.The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 ul of quench solution (final concentrations: 50 mM Tris, pH=7.8, 150 mM NaCl, 0.5 mg/ml BSA, 15 mM EDTA, 48 nM APC/75 nM Streptavidin (Perkin-Elmer Cat. #CR130-100) and 1.5 nM Eu-antibody PY-20 (Perkin-Elmer Cat. #AD0067). After 20 min. incubation at RT plate was read on PHERAstar FS plate reader (BMG Labtech). |
| Affinity data for this assay | |
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