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Updated: Jun 3, 2026

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates
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Phosphorylation assay based on multifunctionalized soluble nanopolymer.

Anton Iliuk1, Juan S Martinez, Mark C Hall

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

Analytical Chemistry
|March 15, 2011
PubMed
Summary
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A new pIMAGO technology enables highly efficient, quantitative protein phosphorylation analysis without phospho-specific antibodies. This sensitive method is crucial for understanding cellular signaling pathways.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Chemical Biology

Background:

  • Quantitative phosphorylation analysis is critical for understanding cellular signal transduction pathways.
  • Current methods often rely on phospho-specific antibodies, which can be costly and have limitations.
  • A need exists for a universal, high-throughput method for phosphorylation assays.

Purpose of the Study:

  • To develop and validate a novel, antibody-free technology for high-throughput quantitative phosphorylation analysis.
  • To assess the sensitivity, specificity, and quantitative accuracy of the new pIMAGO assay.
  • To demonstrate the utility of pIMAGO in various biological applications, including kinase activity and inhibitor screening.

Main Methods:

  • Development of pIMAGO, a water-soluble, nanosize polymer functionalized with titanium(IV) ions for phosphoprotein binding and biotin groups for detection.

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Last Updated: Jun 3, 2026

Nonradioactive Assay to Measure Polynucleotide Phosphorylation of Small Nucleotide Substrates
06:49

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Published on: May 8, 2020

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
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An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Published on: June 6, 2022

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes
09:40

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Published on: September 28, 2018

  • Utilizing enzyme-linked spectrometric detection for quantitative measurement of phosphoprotein levels.
  • Validation using standard phosphoproteins, purified phosphoproteins from cell extracts, in vitro kinase assays, and kinase inhibitor screening.

    • Main Results:

    • The pIMAGO assay demonstrated high sensitivity, detecting as low as 100 pg of phosphoprotein quantitatively.
    • The assay showed high specificity and quantitative accuracy in various biological contexts.
    • Successful application in in vitro kinase assays, kinase inhibitor screening, and measurement of endogenous phosphorylation.

    Conclusions:

    • pIMAGO offers a universal, antibody-free, and quantitative method for global phosphorylation analysis.
    • The technology provides high sensitivity and specificity, advancing the study of cellular signaling.
    • This novel approach facilitates high-throughput screening and analysis of phosphorylation events.