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Method for Determining Gelatinolytic Activity in Tissue Extracts: Real-Time Gelatin Zymography

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The Extracellular Matrix

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1952))

Abstract

To explore the physiological or pathological roles of proteases, it is important to be able to detect and precisely localize them in a tissue, to differentiate between inactive and active forms, as well as to quantify and determine the nature of the enzyme that degrades a given substrate. Here we present a protocol for real-time gelatin zymography that is very useful for the detection of gelatin-degrading proteases in tissue extracts. This method uses fluorescence-labeled gelatin and therefore we also present an easy, fast, and cheap method for labeling gelatin with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF).

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Acknowledgments

We are grateful to Dr. Kristin Andreassen Fenton (Faculty of Health Sciences, Department of Medical Biology, UiT-The Arctic University of Norway, Tromsø, Norway) for the gift of kidney and liver homogenates from a BALB/c mouse and senior engineer Eli Berg (Faculty of Health Sciences, Department of Medical Biology, UiT-The Arctic University of Norway, Tromsø, Norway) for performing the zymography experiments. We also would thank Rod Wolstenholme (Faculty of Health Sciences, UiT-The Arctic University of Norway, Tromsø, Norway) for help with the figure.

Author information

Authors and Affiliations

  1. Faculty of Health Sciences, Department of Medical Biology, UiT—The Arctic University of Norway, Tromsø, Norway

    Elin Hadler-Olsen & Jan-Olof Winberg

  2. Faculty of Health Sciences, Department of Clinical Dentistry, UiT—The Arctic University of Norway, Tromsø, Norway

    Elin Hadler-Olsen

  3. Department of Clinical Pathology, University Hospital of North Norway, Tromsø, Norway

    Elin Hadler-Olsen

Authors
  1. Elin Hadler-Olsen
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  2. Jan-Olof Winberg
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Corresponding authors

Correspondence to Elin Hadler-Olsen or Jan-Olof Winberg .

Editor information

Editors and Affiliations

  1. Department of Medicine and Surgery, University of Insubria, Varese, Italy

    Davide Vigetti

  2. Department of Chemistry, University of Patras, Patras, Greece

    Achilleas D. Theocharis

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Hadler-Olsen, E., Winberg, JO. (2019). Method for Determining Gelatinolytic Activity in Tissue Extracts: Real-Time Gelatin Zymography. In: Vigetti, D., Theocharis, A.D. (eds) The Extracellular Matrix. Methods in Molecular Biology, vol 1952. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9133-4_16

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  • DOI: https://doi.org/10.1007/978-1-4939-9133-4_16

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9132-7

  • Online ISBN: 978-1-4939-9133-4

  • eBook Packages: Springer Protocols

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