| Assay Method Information | |
| | Lck Inhibitory Activity Assay |
| Description: | Tyrosine phosphorylation of Lck was performed by using Z′-LYTE Kinase Assay Kit-Tyr 2 Peptide (Invitrogen) containing the following reagents (Tyr 2 Peptide, Tyr 2 Phospho-Peptide, 5× Kinase Buffer, ATP, Coloring Reagent A, Coloring Buffer, and Stop Reagent) and Lck. The Lck activity was determined by using Fluorescence Resonance Energy Transfer (FRET) method.A dilute solution (5 μL) of the compound of the present invention in dimethylsulfoxide (DMSO; Sigma-Aldrich Co. LLC) was added to a 96-well assay plate. In addition, Peptide/Kinase Buffer composed of DL-dithiothreitol (DTT; 2 mM), Tyr 2 Peptide (2 μM), Kinase Buffer and Lck was added to the assay plate, and the reaction solution was preincubated at 25° C. for 20 minutes. Then, ATP solution (5 μL) composed of adenosine triphosphate (ATP; 45 μM) and Kinase Buffer was added, and the reaction solution was incubated at 25° C. for 1 hour. After incubation, Coloring Solution A (10 μL) composed of Coloring Reagent B and Coloring Buffer was added, and the reaction solution was incubated at 25° C. for 1 hour. Stop Reagent (10 μL) was added to each well such that the enzymatic reaction stopped. The fluorescent coloring of each well was measured on a fluorescent plate reader by using wavelengths of 445 nm and 520 nm. The ratio of phosphorylation was determined by a ratio of coloring at 445 nm to that at 520 nm according to the attached manual. |
| Affinity data for this assay | |
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