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Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae
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DIGE-Based Phosphoproteomic Analysis.

Taras Stasyk1, Lukas Alfons Huber2

  • 1Division of Cell Biology, Biocenter, Innsbruck Medical University, Innrain 80-82, A-6020, Innsbruck, Austria. taras.stasyk@i-med.ac.at.

Methods in Molecular Biology (Clifton, N.J.)
|October 12, 2017
PubMed
Summary

This study details a protocol for detecting phosphoproteins using two-dimensional difference gel electrophoresis (DIGE). Combining DIGE with phosphospecific fluorescent dye staining allows sensitive quantification of differentially regulated phosphoproteins.

Keywords:
2D-DIGEPhosphoproteinPhosphoproteomics

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Area of Science:

  • Proteomics
  • Biochemistry
  • Molecular Biology

Background:

  • Two-dimensional difference gel electrophoresis (DIGE) is a powerful technique for proteome profiling.
  • Detecting post-translational modifications like phosphorylation is crucial for understanding protein function.
  • Existing DIGE protocols may not efficiently or sensitively detect phosphoproteins.

Purpose of the Study:

  • To present a detailed protocol for phosphoprotein detection within DIGE gels.
  • To enhance DIGE analysis by integrating phosphospecific fluorescent staining.
  • To enable sensitive and accurate quantification of differentially regulated phosphoproteins.

Main Methods:

  • A standard two-dimensional difference gel electrophoresis (DIGE) protocol was employed.
  • Subsequent post-staining of the DIGE gel with a phosphospecific fluorescent dye was performed.
  • The combined method allows for fluorescence detection of phosphoproteins in the same gel.

Main Results:

  • The protocol successfully integrates phosphoprotein detection into the DIGE workflow.
  • The combined method provides sensitive detection of phosphoproteins.
  • Accurate quantification of differentially regulated phosphoproteins in biological samples is achievable.

Conclusions:

  • The described protocol enhances DIGE-based proteome profiling by enabling direct phosphoprotein detection.
  • This method offers a sensitive and accurate approach for quantifying changes in phosphoprotein levels.
  • The protocol provides a valuable tool for studying phosphoregulation in biological systems.