| Assay Method Information | |
| | Kinase Assays |
| Description: | The concentration dependent ability of compounds to inhibit human p38α MAPK, p38β MAPK and CK-1δ were done essentially as described in J. Neurosci. 2012, 32, 10201 (herein incorporated by reference in its entirety) and by use of commercially available Millipore Drug Discovery and Development services. IC50 values were calculated by generating a 10-11 point curve and analyzed using nonlinear regression curve fit in GraphPad Prism statistical software. Z-factor (≥0.5) calculation and % CV (<20%) were calculated for each assay. Large-scale kinome screens were done using the commercially available Millipore Profiler test systems (www.millipore.com) that included >291 protein kinases representative of all major kinome branches as well as isoforms of individual families. The NCBI Entrez identifier for each kinase is provided at the vendor site. A hierarchal analysis was done on inhibitors. First, an initial screen was done where each inhibitor was tested at a fixed concentration (20,000 nM) against a panel of protein kinase targets covering the major branches of the kinome, using an ATP concentration for each kinase at or near their apparent Km. Second, preliminary hits from the profiler screen were validated by a follow-up concentration dependent test of the inhibitor to obtain an IC50 value for the inhibitor and a given kinase in order to confirm the hit as positive. Third, kinetic analyses to determine a Ki value were done on confirmed positives with IC50 values<1,000 nM.The commercially available europium competitive active site binding assay (LanthaScreen Eu Kinase Binding Assay, Invitrogen Life Science Technologies, Grand Island, N.Y., USA) that is based on time-resolved fluorescence resonance energy transfer (TR-FRET) technology was used to test for competitive binding to the non-activated form of p38α MAPK. IC50 values were generated from triplicate 12-point curves. Assays were performed in a 384 well plate format (Corning cat No. 3673) in a final volume of 15 μL containing 5 nM kinase (Invitrogen cat No. PV3305), 5 nM tracer (Invitrogen cat No. PV5830), 2 nM antibody (Invitrogen cat No. PV5594). Plates were centrifuged (240 g, 5 min) to mix ingredients, incubation done for 60 min at 25° C., and plates centrifuged (240 g, 5 min) to concentrate reaction mixtures in the plate bottom well. Readings were taken in an En Vision Plate Reader (Perkin Elmer; Waltham Mass., USA) using a dichroic mirror and excitation at wavelength 340 nm (30 nm bandpass) and emission at 665 nm (10 nm bandpass) and 615 nm (10 nm bandpass). Delay time was 100 μs, and integration time 200 μs. The emission ratio was determined by dividing the acceptor/tracer emission (665 nM) by the antibody/donor emission (615 nM). Data were expressed as percent of the maximal binding activity, as determined by the emission ratio, and IC50 values were calculated using GraphPad Prism, version 5.0a, by a nonlinear regression data analysis of log inhibitor concentration versus emission ratio. |
| Affinity data for this assay | |
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