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February 01, 2009 (Vol. 29, No. 3)

Simplification of Kinase Binding Assays

TR-FRET Tools Broaden the Potential of Drug Screening

  • Simple TR-FRET Binding Assay

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    Figure 1. LanthaScreen Eu Kinase Binding Assay

    Invitrogen’s LanthaScreen™ Eu Kinase Binding Assay platform was designed for HTS, lead optimization, and mechanism of inhibition (MOI) studies using kinases in either an active or nonactive conformational state, and to be broadly applicable across the kinome. The assay measures the binding and displacement of an Alexa Fluor® 647 dye–labeled kinase inhibitor (tracer) to a kinase active site.

    Bound tracer is detected by addition of a europium (Eu)-labeled antitag antibody that specifically labels the kinase of interest. When the tracer is bound, fluorescence resonance energy transfer (FRET) occurs between the Eu-labeled antibody and the tracer; when the tracer is displaced by the bound inhibitor, FRET is dramatically reduced (Figure 1).

    In addition to the benefits of flexible target choice, this assay format offers a robust time-resolved–FRET (TR-FRET) readout, simple assay optimization and setup, and the ability to use low concentrations of kinase (in the 0.1 to 5 nM range). Because the tracers are ATP-competitive, the assay will detect any compound that binds to the ATP site. These compounds include Type II inhibitors such as Gleevec, Nexavar, and BIRB-796, which bind to the ATP site, as well as an adjacent allosteric site, exposed in nonactive states of some kinases.

    Using a small set of broad-spectrum tracers, a Eu–anti-GST antibody, and a new high affinity Eu–anti-His antibody, assays have been developed for more than 211 of the 300 kinases in Invitrogen’s portfolio (Figure 2). This broad coverage includes more than 30 kinases for which robust HTS activity assays have been difficult to develop. These newly tractable targets include DDR1, which was recently identified to be a cellular target of Gleevec, as well as CDK8, and TGFBR1 (ALK5) (Figure 3).

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    Figure 2. Kinase coverage
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    Figure 3. IC50 curves for several kinase inhibitors for (A) DDR1, (B) CDK8, and (C) TGFBR1 (ALK5)

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