EMD Biosciences (www.emdbiosciences. com) TruLight Kinase Assays, which do not require antibodies or radioactivity, achieve high-sensitivity by utilizing fluorescence superquenching, a proprietary technology related to fluorescence quenching5.
Fluorescence superquenching occurs for a variety of light-emitting polyelectrolytes6. Small-molecule quenchers can quench the photoluminescence of up to several hundred polymer repeat units. This results in an "amplification" of the quenching potential and the ability to detect fewer quenchers.
TruLight Kinase Assays utilize quenchers that are conjugated to kinase substrate peptides. Fluorescent polymers capable of being superquenched are co-localized with metal ions on microsphere sensors, providing a means for phosphorylated peptides to bind (Figure 1)7. Kinase activity is detected as a decrease in fluorescence when the quencher-labeled phosphorylated peptides bind (Figure 2). All of the assay components have been optimized to meet HTS data requirements.
As a result of superquenching, phosphorylated peptides are detected at lower levels, making TruLight Kinase Assays more sensitive than conventional FRET-based assays (Figure 3). The assays are optimized to exhibit maximum signal change at less than 25% enzyme conversion and exhibit Z values of 0.7 at 10% conversion (Table). This increased sensitivity leads to better detection of weak inhibitors, since IC50 values are lower than those observed at higher enzyme conversion. Additional benefits include cost savings due to lower enzyme requirements and the ability to screen kinases with low turnover.
The assays exhibit ATP tolerances of 100 mM or greater and are compatible with most solvents. Also, they have signal stabilities compatible with HTS. A further advantage for HTS is that ratiometric monitoring at higher wavelengths (~600 nm) can be used with TruLight Kinase Assays to reduce interference from colored or fluorescent compounds.
TruLight Kinase Assays have been developed for Akt1, Aurora A, ERK1/2, p38a, p70S6K, PKA, PKCa, PKCbI/II, PKCe, RSK-2, and Src. The lower limit of detection for these assays varies from 1.2800 picomolar.
Additionally, a Universal Kinase/ Phosphatase Assay is available, whereby the user supplies the quencher-labeled peptide substrate to study any kinase for which a substrate is known. The user determines the substrate, quencher, and reaction conditions needed for the target enzyme.
One adaptation of the TruLight Kinase Assay platform uses protein rather than peptide substrates8. In this mode, the phosphorylated protein substrate binds to the microsphere, and unoccupied sites are "sensed" by subsequent addition of quencher-labeled phospho-peptide. An increase in the fluorescent signal is observed as the kinase reaction proceeds, because the phosphorylated protein substrate blocks the tracer peptide quencher from binding the sensor.
In addition to increasing the versatility of the assays, the ability to utilize protein substrates allows the user to identify novel inhibitors that act via a mechanism that cannot be reproduced using peptide substrates.