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Download Serine / threonine protein kinase assays
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Serine / threonine protein kinase assays introduction Protein kinases are recognised as important drug targets due to the crucial role they play in human diseases such as cancer, inflammatory diseases and, 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 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. optimised for FLEXYTE lifetime reagents robust lifetime readout 1536-well compatible automation-friendly Fig 1. The universal FLEXYTE lifetime Ser/Thr protein kinase assay lifetime application note / serine/threonine protein kinase assays / 2016 Jan v1 page 1 of 2 Ser/Thr kinase Proposed substrate Ser/Thr kinase Proposed substrate AKT1 AKT2 AKT3 AMPK ARK5 ASK1 Aurora A Aurora B Aurora C BrSK1 BrSK2 CAMK1 CaMKIIα CaMKIIβ CaMKIIδ CaMKII γ CaMKIV CDC42 BPA CDC42 BPB CHK1 CHK2 CLK3 DAPK1 DAPK2 DCAMKL2 DMPK DRAK1 DYRK2 eEF-2K GRK5 GRK6 GSK3α GSK3β HIPK2 HIPK3 IKKα IKKβ LKB1 LOK MAP3K8 MAPKAP-K1α MAPKAP-K1β MAPKAP-K2 MAPKAP-K3 MARK1 MELK MINK1 MLCK Mnk2 MSK1 MSK2 MSSK1 MST1 KS1 & Crosstide KS1 KS1 KS3 KS1 KS1 KS2 KS2 KS1 KS1 KS3 KS3 KS3 KS3 KS3 KS3 KS3 KS1 KS1 KS3 KS3 KS1 KS3 KS3 KS3 KS1 KS3 KS1 KS3 KS1 KS1 KS1 KS1 KS1 KS1 KS1 KS1 KS3 KS1 KS1 KS3 KS3 KS3 KS3 KS1 KS3 KS1 KS3 KS3 KS1 KS1 KS1 KS1 MST2 NEK11 NEK2 NEK3 NEK6 NEK7 NLK p38a p70S6K PAK2 PAK3 PAK4 PAK5 PAK6 PAR-1Bα PASK PDK2 PHKγ-2 Pim-1 Pim-2 PKA PKCα PKCβ1 PKCβII PKCδ PKCε PKCγ PKCι PKCµ PKC θ PKC ζ PKG1α PKG1β PLK3 PRAK1 PRK2 ROCK-1 ROCK-II Rsk3 Rsk4 SGK1 SGK2 SGK3 SIK Snk STK33 TBK1 TSSK1 TSSK2 WNK2 WNK3 ZIPK KS1 KS2 KS1 KS1 KS1 KS1 KS1 bespoke KS4 KS1 KS2 KS2 KS2 KS2 KS2 KS2 KS3 KS3 KS3 KS1 KS1 KS2 KS3 KS3 KS3 KS3 KS3 KS3 KS3 KS3 KS3 KS3 KS2 KS2 KS1 KS3 KS3 KS2 KS2 & bespoke KS5 KS3 KS2 KS1 KS1 KS1 KS1 KS1 KS1 KS1 KS3 KS3 KS1 KS1 KS3 For FLEXYTE® support and custom assay design: 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. For ameon® order or support and FLEXYTE® catalogue products: Almac TTP Labtech Ltd 5 Fleming Building, Edinburgh Technopole EH26 0BE, United Kingdom Melbourn Science Park, Melbourn, Hertfordshire SG8 6EE, United Kingdom Tel: +44 (0)28 3839 5717 Email: [email protected] Tel: +44 (0)1763 262 626 Email: [email protected] www.almacgroup.com www.ttplabtech.com lifetime application note / serine/threonine protein kinase assays / 2016 Jan v1 page 2 of 2
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