| Assay Method Information | |
| | LanthaScreen Eu Kinase Binding Assay |
| Description: | Binding of an Alexa Fluor™ conjugate or “tracer” to a kinase is detected by addition of an Eu-labeled anti-tag antibody. Binding of the tracer and antibody to a kinase result in a high degree of FRET, whereas displacement of the tracer with a kinase inhibitor results in a loss of FRET. This assay is carried out by mixing the compound tested with the reagents and reading, no development step is required. Life Technologies' Kinase Tracers are based on ATP-competitive kinase inhibitors, making them suitable for detection of any compounds that bind to the ATP site. Inhibitors that bind the ATP site include both Type I kinase inhibitors, which bind solely to the ATP site, and Type II inhibitors (e.g., Gleevec /Imatinib, Sorafenib, BIRB-796), which bind to both the ATP site and a second site often referred to as the allosteric site. The following protocol is used to carry out this assay: The Test Compounds are screened in 1% DMSO (final) in the well. For 10-point titrations, 3-fold serial dilutions are conducted from the starting concentration. All Kinase/Antibody Mixtures are diluted to a 2× working concentration in the specified kinase buffer. The 4× AlexaFluor labeled Tracer is prepared in Kinase Buffer. Assay Protocol Bar-coded, low volume, white 384-well plate (Greiner Cat. #784207) 1. 160 nL100× Test Compound in 100% DMSO 2. 3.84 μL—Kinase Buffer 3. 8.0 μL2× Kinase/Antibody Mixture 4. 4.0 μL—4× Tracer 5. 30-second plate shake 6. 60-minute incubation at room temperature7. Read on fluorescence plate reader and analyze the data. The following controls are made for each individual kinase and are located on the same plate as the kinase:0% Displacement Control: the maximum Emission Ratio is established by the 0% Displacement Control wells, which do not contain known inhibitor in the reaction and therefore exhibits no displacement of the tracer. 100% Displacement Control: the minimum Emission Ratio is established by the 100% Displacement Control wells, which contain the highest concentration of the known inhibitor used in that assay. Known Inhibitor Control Protocol: a known inhibitor control standard curve, 10-point titration, is run for each individual kinase on the same plate as the kinase to ensure the inhibitor is displaced within an expected IC50 range previously determined. |
| Affinity data for this assay | |
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