Real-Time Protein Kinase Assay
- 15 February 2005
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 77 (7) , 2043-2049
- https://doi.org/10.1021/ac048280e
Abstract
We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.Keywords
This publication has 26 references indexed in Scilit:
- Imaging protein phosphorylation by fluorescence in single living cellsMethods, 2004
- ADP-Specific Sensors Enable Universal Assay of Protein Kinase ActivityChemistry & Biology, 2004
- Development and Validation of a Fluorescence Technology for both Primary and Secondary Screening of Kinases That Facilitates Compound Selectivity and Site-Specific Inhibitor DeterminationASSAY and Drug Development Technologies, 2004
- The Protein Kinase Complement of the Human GenomeScience, 2002
- Protein kinases — the major drug targets of the twenty-first century?Nature Reviews Drug Discovery, 2002
- A Comparison of ALPHAScreen, TR-FRET, and TRF as Assay Methods for FXR Nuclear ReceptorsSLAS Discovery, 2002
- Self-Assembly and Mineralization of Peptide-Amphiphile NanofibersScience, 2001
- Peptide and Protein Library Screening Defines Optimal Substrate Motifs for AKT/PKBJournal of Biological Chemistry, 2000
- Signaling—2000 and BeyondCell, 2000
- A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening AssaysSLAS Discovery, 1999