Purification of large cytosolic proteases for in vitro assays: 20s and 26s proteasomes

Abstract

Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and ef fi ciency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAEion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fl uorogenic substrates and specific inhibitors. © Springer Science+Business Media, LLC 2013.