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Detection of Damage-Activated Metacaspase Activity by Western Blot in Plants

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Plant Proteases and Plant Cell Death

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2447))

Abstract

Metacaspases are cysteine proteases that are present in plants, protists, fungi, and bacteria. Previously, we found that physical damage, e.g., pinching with forceps or grinding on liquid nitrogen of plant tissues, activates Arabidopsis thaliana METACASPASE 4 (AtMCA4). AtMCA4 subsequently cleaves PROPEP1, the precursor pro-protein of the plant elicitor peptide 1 (Pep1). Here, we describe a protein extraction method to detect activation of AtMCA4 by Western blot with antibodies against endogenous AtMCA4 and a PROPEP1-YFP fusion protein. It is important to (1) keep plant tissues at all times on liquid nitrogen prior to protein extraction, and (2) denature the protein lysate as fast as possible, as metacaspase activation ensues quasi immediately because of tissue damage inherent to protein extraction. In theory, this method can serve to detect damage-induced alterations of any protein-of-interest in any organism for which antibodies or fusion proteins are available, and hence, will greatly aid the study of rapid damage-activated proteolysis in the future.

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Acknowledgments

This work was supported by the Research Foundation-Flanders (FWO14/PDO/166 to SS), FWO-Fonds de la Recherche Scientifique (Excellence of Science project no. 30829584 to FVB), and Bijzonder Onderzoeksfonds UGent (BOF19/24 J/008; “DESTINY” Fate and function of metacaspase substrates).

Author information

Authors and Affiliations

  1. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Simon Stael, Álvaro D. Fernández-Fernández & Frank Van Breusegem

  2. VIB-UGent Center for Plant Systems Biology, Ghent, Belgium

    Simon Stael, Álvaro D. Fernández-Fernández & Frank Van Breusegem

  3. Department of Biology, San Diego State University, San Diego, CA, USA

    Luke P. Miller

Authors
  1. Simon Stael
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  2. Luke P. Miller
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  3. Álvaro D. Fernández-Fernández
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  4. Frank Van Breusegem
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Corresponding author

Correspondence to Simon Stael .

Editor information

Editors and Affiliations

  1. Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia

    Marina Klemenčič

  2. VIB-UGent Cntr for Plant Systems Biology, Ghent University, Ghent, Belgium

    Simon Stael

  3. ZEA-3 Analytik, Forschungszentrum Juelich, Juelich, Germany

    Pitter F. Huesgen

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Stael, S., Miller, L.P., Fernández-Fernández, Á.D., Van Breusegem, F. (2022). Detection of Damage-Activated Metacaspase Activity by Western Blot in Plants. In: Klemenčič, M., Stael, S., Huesgen, P.F. (eds) Plant Proteases and Plant Cell Death. Methods in Molecular Biology, vol 2447. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2079-3_11

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  • DOI: https://doi.org/10.1007/978-1-0716-2079-3_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2078-6

  • Online ISBN: 978-1-0716-2079-3

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