serine / threonine protein kinase assays

Protein kinases are recognised as important drug targets due to the crucial role they play in human diseases such as cancer, inflammatory diseases, neuronal and metabolic disorders. Identification of protein kinase modulators has become a common strategy for drug discovery.

The FLEXYTE® lifetime serine / threonine (Ser/Thr) protein kinase assay utilises a unique long lifetime fluorophore coupled to a panel of generic and bespoke peptide substrates to provide a homogeneous, robust, antibody-free and cost-effective screening solution. When analysed with TTP Labtech’s ameon® lifetime reader the resultant platform offers intrinsic tolerance to common assay interferences to deliver unparalleled assay robustness.

assay principle

Ser_Theo_fig1

 

Fig 1. The universal FLEXYTE lifetime Ser/Thr protein kinase assay

benefits of FLEXYTE lifetime assays

A universal FLEXYTE lifetime Ser/Thr protein kinase assay platform has been developed through the design and synthesis of peptide substrates labelled with the long lifetime FLEX17 fluorophore. Phosphorylation of the substrate is detected through addition of a small molecule iron(III) chelator which complexes the phosphate group and reduces the fluorophore lifetime. Consequently, the reported lifetime of the reaction correlates with the level of phosphorylation of the substrate.

Utilisation of a long lifetime FLEX17 fluorophore enables discrimination of substrate and product from fluorescent interfering compounds whose lifetimes are commonly below 2 nanoseconds, and also provides a stable, defined assay window upon which the assays can be built in 384- or 1536-well microplates.

ameon® lifetime reader

TTP Labtech’s ameon lifetime reader meets the challenge of accelerating the drug discovery process by integrating easily into screening workflows and offering a reliable fluorescence lifetime readout. The instrument applies a proprietary data recording technology to perform real-time decay curve capture and delivers precise lifetime calculation at high throughput.

specific enzyme list

KS1, 2 and 3 are three generic substrates which enable assay configuration for a broad panel of Ser/ Thr kinases.

Bespoke FLEX17-labelled peptides tailored for a specific kinase of interest can be synthesised upon request.

Ser_Theo_table

View our serine / threonine protein kinase assays app note

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