Published in
BioTechniques Protocol Guide 2007
(p.47)
Protocol Abstract
Dysregulation of kinase activity plays a key role in many human diseases, making kinase inhibitors highly attractive drug candidates. PerkinElmer has a variety of luminescence, fluorescence and radioactive assay platforms for cell-based and biochemical kinase screening, profiling and validation. The selection of a kinase assay platform can depend upon the size of the kinase substrate, the throughput requirements, assay format, and the availability of a phospho-substrate antibody. PerkinElmer's LANCE® Ultra, with Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) technology, is ideally suited for ultra high-throughput kinase inhibitor screening and validation.
LANCE Ultra reagents include a series of europium chelate-labeled anti-phospho-substrate antibodies and several kinase peptide substrates directly labeled with the new proprietary dye, ULight™, which is a low molecular weight red-shifted dye. The direct labeling of the peptide substrate in combination with the use of the ULight dye, make LANCE Ultra TR-FRET assays highly efficient with less steric hindrance, with excellent Z’ values and prolonged assay stability when compared to traditional TR-FRET assays that utilize larger, less efficient dye combinations.
Figure 1. (Click to enlarge)
In this protocol we describe the development of a homogeneous kinase activity assay by monitoring the phosphorylation of the MBP peptide substrate by an ERK1 kinase. The MBP peptide is directly labeled with ULight, and the anti-phospho-MBP antibody is labeled with europium.
Figure 2. (Click to enlarge)
Protocol Summary:
Easy set up of a kinase activity assay utilizing highly efficient TR-FRET dyes: Europium and ULight
Assay is homogeneous and applicable for ultra high-throughput
The optimized assay developed using LANCE Ultra reagents shows robust performance suitable for HTS, as demonstrated by a Z’ value of 0.83.
Simplicity of use and minimal interference with screening compounds make LANCE Ultra the platform of choice for HTS kinase assays
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