Overcoming compound interference in fluorescence polarization-based kinase assays using far-red tracers

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Kinase-mediated phosphorylation regulates crucial biological processes like cell growth and apoptosis.
• Targeting kinases with small-molecule inhibitors is a key therapeutic strategy for diseases.
• Traditional kinase assays (ELISA, radioactive) have limitations, driving interest in homogenous fluorescent assays.

Purpose of the Study:

• To develop a robust homogenous fluorescent assay for kinase activity.
• To overcome interference issues common in fluorescent assays, such as compound autofluorescence and light scatter.
• To present a far-red fluorescence polarization assay format that enhances reliability and assay window.

Main Methods:

• Development of the PanVera PolarScreen far-red fluorescence polarization assay.
• Utilized a far-red fluorescent tracer to mitigate interference.
• Compared performance against existing fluorescein-based and Cy5-based assays.

Main Results:

• The far-red fluorescence polarization assay effectively reduces interference from autofluorescent compounds.
• The assay demonstrates reduced sensitivity to light scatter compared to lower-wavelength probes.
• The new format provides a larger assay window than the popular Cy5 far-red fluorophore.

Conclusions:

• The PanVera PolarScreen far-red fluorescence polarization assay offers a reliable method for kinase activity screening.
• This assay format mitigates common interferences, enhancing drug discovery efforts.
• The use of far-red probes is a viable strategy for improving homogenous fluorescent assays.