Protocol for the analysis of proteasome activity, assembly state, and composition in fission yeast extracts using native gels.

Abstract

The proteasome is a macromolecular complex responsible for degrading short-lived or damaged proteins. Proteasome analysis presents challenges due to its high molecular weight and low stability. Here, we present a protocol to assess proteasome activity, assembly, and composition in Schizosaccharomyces pombe. We describe steps for lysate preparation and native electrophoresis to separate proteasome complexes. We then detail procedures for detecting proteasome activity using fluorescent substrates, fluorescently-tagged native proteins, and non-tagged proteasome components by immunodetection in a single gel.