Purification and characterization of different proteasome species from mammalian cells

Proteasomes are heterogeneous in forms and functions, but how the equilibrium among the 20S, 26S, and 30S proteasomes is achieved and altered is elusive. Here, we present a protocol for purifying and characterizing proteasome species. We describe steps for generating stable cell lines; affinity purifying the proteasome species; and characterizing them through native PAGE, activity assay, size-exclusion chromatography, and mass spectrometry. These standardized methods may contribute to biochemical studies of cellular proteasomes under both physiological and pathological conditions. For complete details on the use and execution of this protocol, please refer to Choi et al. (2023).¹.