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Native Gel Approaches in Studying Proteasome Assembly and Chaperones

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The Ubiquitin Proteasome System

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

Abstract

Proteasomes are complex molecular machines that consist of 66 subunits. The assembly of these complexes is highly coordinated in a process that requires at least ten proteasome-specific molecular chaperones. One of the challenges in studying assembly intermediates is their relatively low abundance as compared to the proteasome holoenzyme. Therefore, superior separating techniques are crucial for analyses of proteasomal complexes in general and studies in the assembly in particular. For this reason, native gel analyses have been abundantly used in studying proteasomes, as they provide a high resolution. Native gels are very versatile and can be used in various combinatorial approaches. In this chapter, we outline two approaches to prepare samples for native gels. The first is a yeast cryogrinding method and the second a core particle (CP)-base reconstitution approach. We describe the native gel electrophoresis, as well as various downstream analyses, including 2D native-SDS-PAGE. These techniques and approaches can all be used, often in parallel, to gain a variety of information about activity and composition of the complexes separated by native gel. The potential combined approaches are discussed in this review.

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Acknowledgments

Many of these techniques described above have been developed or optimized and tweaked by many experts in the field, and the power of their use has been displayed in numerous excellent papers. We apologize for not being able to provide a comprehensive reference to all those contributions in this publication. This work was supported in part by grants to J.R. from NIH (R01-GM118660 and R15-GM112142).

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Authors and Affiliations

  1. Division of Biology, Kansas State University, Manhattan, KS, USA

    Jeroen Roelofs, Anjana Suppahia & Kenrick A. Waite

  2. Department of Molecular, Cellular, and Developmental Biology, University of Colorado Boulder, Boulder, CO, USA

    Soyeon Park

Authors
  1. Jeroen Roelofs
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  2. Anjana Suppahia
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  3. Kenrick A. Waite
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  4. Soyeon Park
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Corresponding authors

Correspondence to Jeroen Roelofs or Soyeon Park .

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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Roelofs, J., Suppahia, A., Waite, K.A., Park, S. (2018). Native Gel Approaches in Studying Proteasome Assembly and Chaperones. 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_16

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  • DOI: https://doi.org/10.1007/978-1-4939-8706-1_16

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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

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