Measuring Proteasome Activity in Different Subcellular Compartments of the Rat Brain

Abstract

Source: McFadden, T., et al. Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain. J. Vis. Exp. (2019).

This video demonstrates how to measure proteasome activity in different subcellular compartments of the lateral amygdala from fear-conditioned and control rat brains using a fluorogenic peptide assay. The proteasome cleaves the fluorogenic peptide substrate, releasing fluorescent molecules, which are quantified using a plate reader to assess proteasome activity in the nuclear and synaptic protein fractions. Increased nuclear activity suggests changes in gene expression, while reduced synaptic activity indicates protein stabilization necessary for long-term memory maintenance.

Protocol

1. Proteasome Activity Assay

NOTE: Proteasome activity can be measured in homogenized brain tissue using a slightly modified version of the 20S Proteasome Activity Kit. This assay does not directly measure the activity of complete 26S proteasome complexes. Rather, it measures the activity of the 20S core, meaning it can only serve as a proxy to understand the activity of the core itself as opposed to the entire 26S proteasome complex. The success of this assay declines with repeated freeze-thaw cycles and/or increasing levels of detergents, particularly ionic, and requires the use of a plate reader with a 360....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
20S Proteasome Activity Kit	Millipore Sigma	APT280	Other vendors carry different versions
ATP	Fisher	FERR1441	Various other vendors
BioTek Synergy H1 plate reader	BioTek	VATECHH1MT3	Other vendors carry different versions
Protease Inhibitor	Millipore Sigma	P8340	Various other vendors
DMSO	DMSO	D8418	Various other vendors