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Live Cell Imaging and Profiling of Cysteine Cathepsin Activity Using a Quenched Activity-Based Probe

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Activity-Based Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1491))

Abstract

Since protease activity is highly regulated by structural and environmental influences, the abundance of a protease often does not directly correlate with its activity. Because in most of the cases it is the activity of a protease that gives rise to its biological relevance, tools to report on this activity are of great value to the research community. Activity-based probes (ABPs) are small molecule tools that allow for the monitoring and profiling of protease activities in complex biological systems. The class of fluorescent quenched ABPs (qABPs), being intrinsically “dark” and only emitting fluorescence after reaction with the target protease, are ideally suited for imaging techniques such as small animal noninvasive fluorescence imaging and live cell fluorescence microscopy. An additional powerful characteristic of qABPs is their covalent and irreversible modification of the labeled protease, enabling in-depth target characterization. Here we describe the synthesis of a pan-cysteine cathepsin qABP BMV109 and the application of this probe to live cell fluorescence imaging and fluorescent SDS-PAGE cysteine cathepsin activity profiling.

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Author information

Authors and Affiliations

  1. Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 RoyalParade, Parkville, VIC, Australia

    Laura E. Edgington-Mitchell

  2. Departments of Pathology, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA, 94305-5324, USA

    Matthew Bogyo

  3. Cancer Biology Program, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA, 94305-5324, USA

    Matthew Bogyo

  4. Department of Microbiology and Immunology, Stanford School of Medicine, 300 Pasteur Drive, Stanford, CA, 94305-5324, USA

    Matthew Bogyo

  5. Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, RadboudUMC, Geert Grooteplein 26/28, 6525, GA Nijmegen, The Netherlands

    Martijn Verdoes

Authors
  1. Laura E. Edgington-Mitchell
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  2. Matthew Bogyo
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  3. Martijn Verdoes
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Corresponding author

Correspondence to Martijn Verdoes .

Editor information

Editors and Affiliations

  1. Bio-Organic Synthesis Group Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands

    Herman S. Overkleeft

  2. Bio-Organic Synthesis Group Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands

    Bogdan I. Florea

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Edgington-Mitchell, L.E., Bogyo, M., Verdoes, M. (2017). Live Cell Imaging and Profiling of Cysteine Cathepsin Activity Using a Quenched Activity-Based Probe. In: Overkleeft, H., Florea, B. (eds) Activity-Based Proteomics. Methods in Molecular Biology, vol 1491. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6439-0_11

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  • DOI: https://doi.org/10.1007/978-1-4939-6439-0_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6437-6

  • Online ISBN: 978-1-4939-6439-0

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