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Methods to Rapidly Prepare Mammalian 26S Proteasomes for Biochemical Analysis

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The Ubiquitin Proteasome System

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1844))

Abstract

Rapid, gentle isolation of 26S proteasomes from cells or tissues is an essential step for studies of the changes in proteasome activity and composition that can occur under different physiological or pathological conditions and in response to pharmacological agents. We present here three different approaches to affinity purify or to prepare proteasome-rich cell fractions. The first method uses affinity tags fused to proteasome subunits and has been useful in several cell lines for studies of proteasome structure by cryo-electron microscopy and composition by mass spectrometry. A second method uses the proteasome’s affinity for a ubiquitin-like (UBL) domain and can be used to purify these particles from any cell or tissue. This method does not require expression of a tagged subunit and has proven to be very useful to investigate how proteasomal activity changes in different physiological states (e.g., fasting or aging), with neurodegenerative diseases, and with drugs or hormones that cause subunit phosphorylation. A third, simple method that is based on the 26S proteasome’s high molecular weight (about 2.5 MDa) concentrates these particles greatly by differential centrifugation. This method maintains the association of proteasomes with ubiquitin (Ub) conjugates and many other loosely associated regulatory proteins and is useful to study changes in proteasome composition under different conditions.

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Acknowledgments

The methods described here were developed through research support to our laboratory from the NIH-NIGMS (R01 GM51923), Cure Alzheimer’s Fund, Muscular Dystrophy Association (MDA-419143), and Project A.L.S (2015-06).

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Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Chueh-Ling Kuo, Galen Andrew Collins & Alfred L. Goldberg

Authors
  1. Chueh-Ling Kuo
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  2. Galen Andrew Collins
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  3. Alfred L. Goldberg
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Corresponding author

Correspondence to Alfred L. Goldberg .

Editor information

Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada

    Thibault Mayor

  2. Department of Chemistry and Biochemistry, University of Nevada Las Vegas, Las Vegas, NV, USA

    Gary Kleiger

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Kuo, CL., Collins, G.A., Goldberg, A.L. (2018). Methods to Rapidly Prepare Mammalian 26S Proteasomes for Biochemical Analysis. In: Mayor, T., Kleiger, G. (eds) The Ubiquitin Proteasome System. Methods in Molecular Biology, vol 1844. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8706-1_18

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  • DOI: https://doi.org/10.1007/978-1-4939-8706-1_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8705-4

  • Online ISBN: 978-1-4939-8706-1

  • eBook Packages: Springer Protocols

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