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Related Experiment Videos

Real-time protein kinase assay.

Hongye Sun1, Karen E Low, Sam Woo

  • 1Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

Analytical Chemistry
|April 2, 2005
PubMed
Summary
This summary is machine-generated.

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A new real-time fluorogenic kinase assay uses self-assembling peptide substrates. Phosphorylation triggers a 4-6 fold fluorescence increase, enabling robust kinase activity monitoring and inhibitor screening.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Assay Development

Background:

  • Protein kinases are crucial drug targets, necessitating sensitive and real-time assay technologies.
  • Existing fluorogenic assays have limitations, including fluorophore proximity to the phosphorylation site.

Purpose of the Study:

  • To develop a novel, real-time fluorogenic kinase assay with enhanced sensitivity and versatility.
  • To demonstrate the assay's applicability for kinase inhibitor screening and high-throughput screening (HTS).

Main Methods:

  • Peptide substrates synthesized with a fluorescent dye and hydrocarbon tail self-assemble into micelles, quenching fluorescence.
  • Phosphorylation induces micelle reorganization, leading to a significant fluorescence increase.
  • Dynamic light scattering and cryo-electron microscopy validated structural changes.

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Main Results:

  • The assay demonstrated a 4-6 fold fluorescence increase upon phosphorylation.
  • Successful development of assays for multiple kinases, including PKA, PKC, and src-family kinases.
  • Consistent IC(50) values for inhibitors and successful HTS using the LOPAC library.

Conclusions:

  • This novel fluorogenic assay provides a robust and sensitive method for real-time kinase activity detection.
  • The assay's design overcomes limitations of previous systems, allowing fluorophore placement distant from the phosphorylation site.
  • The technology is suitable for kinase inhibitor profiling and high-throughput screening applications.