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Development and Optimization of Kinase Activity Assays using LANCEĀ® Ultra (TR-FRET) Reagents
Sponsored,vendor-submitted protocol   Sponsored by PerkinElmer    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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