| Assay Method Information | |
| | Inhibition Tests on JAK-1, JAK-2 and JAK-3 Kinases |
| Description: | In Kinase Reaction, JAK-1, JAK-2 or JAK-3 may transfer γ-phosphoric acid in ATP onto a single tyrosine residue of a peptide substrate; and there were JAK-1, JAK-2 or JAK-3 inhibitors in the system, γ-phosphoric acid groups on ATP would be not transferred onto the peptide substrate, thus resulting in the failure of phosphorylation. An evaluation experiment was designed based on the principle, a peptide substrate was designed with kinase phosphorylation sites, namely, protein restriction enzyme cutting sites; and both ends thereof were respectively connected with 2 fluorophores as a donor and a receptor respectively; if the kinase kept activity in the system, a γ-phosphonic acid group was transferred onto restriction enzyme cutting sites of the substrate, so that the γ-phosphonic acid group would not be cut and separated into two segments by a protease; moreover, under the stimulation of a laser having a specific wavelength, energy from a segment of fluorescence would be transferred onto another end of fluorophores, thus emitting energy. Otherwise, after kinase activity was inhibited, phosphoric acid groups would be not transferred, and restriction enzyme cutting sites in the substrate would be cut by a kinase in the system to separate the substrate into two segments, thereby no energy transfer of fluorescence occurred. Based on this, the activity of the kinase was evaluated. In this experiment, a 10 μl kinase reaction system was selected, firstly, 2.5 μl kinase (concentration: 1 nM), 2.5 μl peptide substrate (concentration: 2 μM), 2.5 μl ATP (concentration: 10 PM) and 2.5 μl compound were added to each system for reaction for 1 h at room temperature, and then a 5 μl test solution was added for reaction for 1 h at room temperature, then a 5 μl stop buffer was added. A microplate reader (Synergy H1, BioTek, USA) was used to measure fluorescence intensity (emission intensity of coumarin at 445 nm and emission intensity of fluorescein at 520 nm were detected under the stimulation of 400 nm). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |