Real-time protein kinase assay

被引：24

作者：

Sun, HY ^{[1
]}

Low, KE ^{[1
]}

Woo, S ^{[1
]}

Noble, RL ^{[1
]}

Graham, RJ ^{[1
]}

Connaughton, SS ^{[1
]}

Gee, MA ^{[1
]}

Lee, LG ^{[1
]}

机构：

[1] Appl Biosyst Inc, Foster City, CA 94404 USA

来源：

ANALYTICAL CHEMISTRY | 2005年 / 77卷 / 07期

关键词：

D O I：

10.1021/ac048280e

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

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. IC50 values of inhibitors for PKC beta II 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.

引用

页码：2043 / 2049

页数：7

共 32 条

[1] Detection of p56lck kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384-and 1536-well format [J].