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Purification of Large Cytosolic Proteases for In Vitro Assays: 20S and 26S Proteasomes

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Antigen Processing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1988))

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Abstract

Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and efficiency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAE-ion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fluorogenic substrates and specific inhibitors.

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft TE599/1-1, TE599/2-1 and the Forschungszentrum Immuntherapie (FZI CF7) of the University Mainz.

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Authors and Affiliations

  1. Institute for Immunology, University Medical Center of the Johannes Gutenberg-Universität Mainz, Langenbeckstr. 1, Mainz, Germany

    Stefan Tenzer & Hansjörg Schild

Authors
  1. Stefan Tenzer
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  2. Hansjörg Schild
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Corresponding author

Correspondence to Stefan Tenzer .

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Editors and Affiliations

  1. INSERM U1151, Université Paris Descartes, Hopital Necker-Enfants Malades, Paris, France

    Peter van Endert

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Tenzer, S., Schild, H. (2019). Purification of Large Cytosolic Proteases for In Vitro Assays: 20S and 26S Proteasomes. In: van Endert, P. (eds) Antigen Processing. Methods in Molecular Biology, vol 1988. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9450-2_1

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  • DOI: https://doi.org/10.1007/978-1-4939-9450-2_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9449-6

  • Online ISBN: 978-1-4939-9450-2

  • eBook Packages: Springer Protocols

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