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Related Experiment Video

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Assaying Protein Kinase Activity with Radiolabeled ATP
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Efficient PKC inhibitor screening achieved using a quantitative CE-LIF assay.

Binh Thanh Nguyen1,2, Min Park3, Jae-Chul Pyun4

  • 1Molecular Recognition Research Center, Korea Institute of Science and Technology (KIST), Seoul, Korea.

Electrophoresis
|October 27, 2016
PubMed
Summary

A new assay quantifies protein kinase C delta (PKCδ) activity using capillary electrophoresis. This method is adaptable for screening kinase inhibitors in drug discovery.

Keywords:
CE-LIFKinaseProtein Kinase CScreening of kinase inhibitor

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Pharmacology

Background:

  • Protein kinase C delta (PKCδ) plays a role in various cellular processes.
  • Accurate measurement of PKCδ activity is crucial for understanding its function and for drug discovery.
  • Existing methods for kinase activity assays may have limitations in adaptability.

Purpose of the Study:

  • To develop and validate a novel assay for quantifying PKCδ activity.
  • To utilize capillary electrophoresis with laser-induced fluorescence detection for sensitive detection of a synthetic substrate.
  • To assess the potential of this assay for high-throughput screening of kinase inhibitors.

Main Methods:

  • Development of a capillary electrophoresis (CE) method with laser-induced fluorescence (LIF) detection.
  • Use of fluorescein isothiocyanate-labeled peptides (F-ERK and phosphorylated P-F-ERK) as synthetic substrates.
  • Validation of the assay for sensitivity (LOD/LOQ), reproducibility, and accuracy.
  • In vitro testing of PKCδ activity in human gastric cancer cells (MKN-1) and evaluation of inhibitor efficacy (IC50 values).

Main Results:

  • The CE-LIF assay demonstrated good sensitivity with low limits of detection and quantification for both substrate forms.
  • High correlation coefficients (approx. 0.99) and acceptable reproducibility and accuracy were achieved.
  • The assay successfully determined IC50 values for known PKCδ inhibitors, showing good agreement with literature values.
  • The developed assay showed greater adaptability to different enzyme isoforms compared to a commercial kit.

Conclusions:

  • A robust and sensitive CE-LIF assay for PKCδ activity has been established.
  • This assay is suitable for evaluating the potency of PKCδ inhibitors.
  • The method's adaptability makes it a valuable tool for drug discovery programs, particularly for high-throughput screening of kinase inhibitors.