Native Gel Approaches in Studying Proteasome Assembly and Chaperones

Methods in Molecular Biology
Jeroen RoelofsSoyeon Park

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.

Citations

Mar 15, 2019·Pharmacological Reviews·Tiffany A Thibaudeau, David M Smith
Oct 24, 2020·Scientific Reports·Kenrick A WaiteJeroen Roelofs
Feb 28, 2021·The Journal of Biological Chemistry·Alicia BurrisJeroen Roelofs
Apr 14, 2021·Nature Structural & Molecular Biology·Helena M SchnellJohn Hanna

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