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Anal Chem. 2011 Apr 1;83(7):2767-74. doi: 10.1021/ac2000708. Epub 2011 Mar 11.

Phosphorylation assay based on multifunctionalized soluble nanopolymer.

Author information

1
Department of Biochemistry and Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907, USA.

Abstract

Quantitative phosphorylation analysis is essential to understanding cellular signal transductions. Here we present a novel technology for the highly efficient assay of protein phosphorylation in high-throughput format without the use of phospho-specific antibodies. The technique is based on a water-soluble, nanosize polymer, termed pIMAGO, that is multifunctionalized with titanium(IV) ions for specific binding to phosphoproteins and with biotin groups that allow for enzyme-linked spectrometric detection. The sensitivity, specificity, and quantitative nature of pIMAGO for phosphorylation assays were examined with standard phosphoproteins and with purified phosphoproteins from whole cell extracts. As low as 100 pg of phosphoprotein can be measured quantitatively with the pIMAGO chemiluminescence assay. The pIMAGO assay was applied to an in vitro kinase assay, kinase inhibitor screening, and measurement of endogenous phosphorylation events. The technique provides a universal, quantitative method for global phosphorylation analysis with high sensitivity and specificity.

PMID:
21395237
PMCID:
PMC3069141
DOI:
10.1021/ac2000708
[Indexed for MEDLINE]
Free PMC Article

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