Real-time protein kinase assay
| Autor: | Ronald J. Graham, Linda G. Lee, Sonia S. Connaughton, Karen E. Low, Melissa Gee, Hongye Sun, Richard L. Noble, Sam L. Woo |
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| Rok vydání: | 2005 |
| Předmět: |
Fluorophore
p38 mitogen-activated protein kinases Drug Evaluation Preclinical Chemistry Techniques Analytical Fluorescence Analytical Chemistry Substrate Specificity chemistry.chemical_compound Adenosine Triphosphate Combinatorial Chemistry Techniques Scattering Radiation Phosphorylation Protein kinase A Protein Kinase Inhibitors Protein kinase C Micelles Protein Kinase C Quenching (fluorescence) Kinase Phosphopeptide Cryoelectron Microscopy Titrimetry Reproducibility of Results chemistry Biochemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Protein Kinases |
| Zdroj: | Analytical chemistry. 77(7) |
| ISSN: | 0003-2700 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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