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Measurements of the Intracellular Stability of CPPs

  • Ivo R. Ruttekolk
  • Wouter P. R. Verdurmen
  • Yi-Da Chung
  • Roland Brock
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 683)

Abstract

Nowadays, the analysis of the uptake and intracellular distribution of cell-penetrating peptides mostly relies on fluorescence microscopy, using fluorescently labeled CPP analogs. However, fluorescence microscopy does not reveal to which degree fluorescence reflects the intact peptide or only breakdown products. Here, we introduce fluorescence correlation spectroscopy (FCS) as a powerful method to address peptide stability in cells and cell lysates. Measurements in lysates of cells incubated with peptide yield information on degradation of the total cellular peptide content. In combination with protease inhibitors, such measurements enable conclusions on trafficking pathways. Intracellular FCS measurements provide direct information on peptide degradation and association with cellular structures in intact cells.

Key words

Cell-penetrating peptide Fluorescence correlation spectroscopy Proteolytic stability Fluorescence Microscopy Peptide degradation 

Notes

Acknowledgments

The authors acknowledge financial support from the Volkswagen-Foundation (Nachwuchsgruppen an Universitäten, I/77 472) and from the Radboud University Nijmegen Medical Centre to WPRV.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Ivo R. Ruttekolk
    • 1
  • Wouter P. R. Verdurmen
    • 1
  • Yi-Da Chung
    • 1
  • Roland Brock
    • 1
  1. 1.Department of Biochemistry, Nijmegen Centre for Molecular Life SciencesRadboud University Nijmegen Medical CentreNijmegenThe Netherlands

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