Synthesis and Application of Activity-Based Probes for Proteases

  • Tim Van Kersavond
  • Minh T. N. Nguyen
  • Steven H. L. VerhelstEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1574)


The detection, visualization, and identification of active proteases can be facilitated by activity-based probes, which covalently bind to a catalytic residue of the target protease. The synthesis of activity-based probes can be challenging. We here outline a simple protocol for probe synthesis based on standard solid phase peptide synthesis followed by capping of the N-terminus with a reactive electrophile as a warhead. The applicability of the probes is illustrated by labeling cysteine proteases in cell and tissue lysates with Western blotting or fluorescence scanning as a readout.

Key words

Activity-based probes Activity profiling Chemical proteomics Click chemistry Proteases Solid phase peptide synthesis 



We acknowledge funding by the Deutsche Forschungsgemeinschaft DFG and the Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen.


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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Tim Van Kersavond
    • 1
  • Minh T. N. Nguyen
    • 1
  • Steven H. L. Verhelst
    • 1
    • 2
    Email author
  1. 1.Leibniz Institute for Analytical Sciences ISAS, e.v.DortmundGermany
  2. 2.Department of Cellular and Molecular MedicineKU Leuven—University of LeuvenLeuvenBelgium

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