| Assay Method Information | |
| | JAK3 Activity Inhibitory Assay |
| Description: | In the kinase reactions, fused proteins (6His tag-fused hJAK3 kinase domain (aa781-end)) which were coexpressed in Sf21 cells and purified by Ni2+/NTA agarose were used. The kinase reactions were initiated by the addition of the following solutions of (a) to (c) to 96-well half-area white plates (plates, Corning Incorporated 3642).(a) 5 gmol/L TK substrate-biotin (cisbio) diluted by kinase buffer (50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.0)), 0.02% sodium azide, 0.1 mmol/L sodium vanadate, 5 mmol/L magnesium chloride, 1 mmol/L dithiothreitol, 0.01% bovine serum albumin), 25 μmol/L ATP, 250 nmol/L Supplement Enzymatic buffer (cisbio) solution: 10 μL/well(b) Test-article solution prepared by using kinase buffer containing 5% dimethylsulfoxide: 10 μL/well(c) 33 ng/mL hJAK3 enzyme diluted by kinase buffer: 30 μL/wellA well in which ATP was not added was set out as a blank well.Plates were let stand at room temperature for 10 minutes from starting reactions.To the plates were added 50 μL/well of a buffer for detection containing TK-Antibody-Cryptate (5 tests/50 μL) and streptoavidine-addition XL665 (62.5 nmol/L) reagent (50 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.0), 20 mM EDTA, 800 mmol/L potassium fluoride, 0.1% bovine serum albumin).One hour after the addition of the buffer for detection, fluorescence counts of each well were measured by a fluorescence microplate reader. Specifically, fluorescence counts in 620 nm excited in 337 nm, and fluorescence counts in 665 nm excited by fluorescence in 620 nm were measured.Ratio of each well was calculated from measured fluorescence counts (fluorescence counts in 665 nm/fluorescence counts in 620 nm×10000).Data were obtained by deducting the average Ratio of a blank well from Ratio of each well. IC50 values of test articles were calculated from % of control values of 2 doses before as well as after 50% in 100% as % of a control value of a solvent control. % Inhibition of either 0.1 μmol/L or 1 μmol/L (100-% of control values) was also calculated. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |