Fluorescence Detection Assays

The one-step, extremely basic fluorescence-based kinase assay is a great substitute for radioactive methods and is especially useful for high-throughput screening of drugs for kinase inhibitors. Creative Biolabs provides kinase drug discovery services through the use of fluorescence detection assays, such as those based on fluorescence polarization, fluorescence resonance energy transfer, and fluorescence intensity measurement.

Fluorescence Detection Assays for Kinases Drug Discovery

• Fluorescence polarization-based kinase assay

Fluorescence polarization (FP), a ratiometric technique, depends on the disparities in rotational properties between large and small molecules. The FP-based kinase assay is a reasonably simple one-step method for high-throughput screening of compounds for tyrosine kinase inhibitors. Fluorescence photometry measurement is independent of both light intensity and fluorophore concentration. However, several of the reaction components' intrinsic fluorescence may make the FP test difficult. These interferences can be prevented by labeling the fluorescent indicator with different excitation and emission wavelengths, like Texas red or rhodamine.

• Fluorescence resonance energy transfer-based kinase assay

The technique of FRET involves the transfer of excited state energy from an excited donor fluorophore to an acceptor fluorophore without the need for radiation. A donor fluorophore is coupled to an antibody in a FRET-based kinase assay, which specifically binds to a phosphorylated reaction product tagged with an appropriate acceptor fluorophore. Resonant energy transfer is possible because of the proximity that antibody-mediated contact creates between the donor and acceptor.

• Fluorescence intensity measurement-based protein kinase assay

In the kinase assay, the phosphospecific antibody or peptide substrate was labeled using various fluorescent probes. A microarray, a high-throughput platform for numerous bioanalytical investigations, was employed to evaluate protein kinase activity when high-throughput assays were needed. Peptide substrates were immobilized on the solid supports; an addressable fluorescent spot is produced when phosphorylation by kinase causes a fluorescent phospho-specific antibody to attach to the appropriate phosphopeptides.

Fig.1. Fluorescence detection assays.¹

Advantages of Fluorescence Detection Assays

• The fluorescence detection assays have high sensitivity.
• The fluorescence detection assays are non-radioactive.
• The fluorescence detection assays make the process of detection easier because they work with some automated devices.

In the field of kinase drug discovery, fluorescence detection assays have established themselves as a reliable technique for both analytical and high-throughput applications. Assays for fluorescence detection have mostly been employed in screening, although they can also be utilized in compound profiling and target validation. For almost all of the main target classes used in drug discovery, including GPCRs, kinases, proteases, nuclear receptors, and phosphatases, fluorescence detection assays have been created. Because of advancements in instrumentation and biology, drug discovery applications have progressed quickly. There are numerous primary screening assays available on the market, together with the protein targets. Please contact us for more information about our fluorescence detection assays for kinase drug discovery and our kinase-related products.

Reference

1. Li, YongJin, WeiHong Xie, and GuiJie Fang. "Fluorescence detection techniques for protein kinase assay." Analytical and bioanalytical chemistry 390 (2008): 2049-2057.