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Sci. STKE, 2 November 2004
Vol. 2004, Issue 257, p. tw393
[DOI: 10.1126/stke.2572004tw393]

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METHODS Supersensitive High-Throughput Phosphorylation Screen

Rininsland et al. describe a highly sensitive method for performing high-throughput screening for kinase and phosphatase activity using Ga3+-mediated fluorescence superquenching. Because protein phosphorylation is central to the regulation of cellular activity, the enzymes that regulate this posttranslational modification are potential targets for drug therapy. Rininsland et al. created a sensor platform from a modified anionic polyelectrolyte poly(p-phenylene-ethynylene) (PPE) derivative, the fluorescence of which can be selectively quenched by rhodamine-labeled phosphopeptides in the presence of Ga3+, but not by the unphosphorylated peptides. Detection of native phosphopeptides was achieved using a competition assay, whereby the addition of the non-dye-labeled phosphopeptide blocked the quenching by the dye-conjugated phosphopeptide. Proof of principle was demonstrated by measuring the activities of several known protein kinases and phosphatases in the presence and absence of pharmacological inhibitors.

F. Rininsland, W. Xia, S. Wittenburg, X. Shi, C. Stankewicz, K. Achyuthan, D. McBranch, D. Whitten, Metal ion-mediated polymer superquenching for highly sensitive detection of kinase and phosphatase activities. Proc. Natl. Acad. Sci. U.S.A. 101, 15295-15300 (2004). [Abstract] [Full Text]

Citation: Supersensitive High-Throughput Phosphorylation Screen. Sci. STKE 2004, tw393 (2004).


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