Fractionation of Native Protein Complexes from Mammalian Cells to Determine the Differential Proteasome Activity and Abundance

Ana Paula, Zen Petisco Fiore, Christine Vogel

Research output: Contribution to journalArticlepeer-review


Eukaryotic cells have different types of proteasomes that differ in size. The smallest proteolytically active particle is the 20S proteasome, which degrades damaged and oxidized proteins; the most common larger particle is the 26S proteasome, which degrades ubiquitylated proteins. The 26S proteasome is formed by a 20S particle capped with one or two regulatory particles, named 19S. While proteasome particles function in the cytoplasm, endoplasmic reticulum, and nucleus, our understanding of their abundance and activity in different cellular compartments is still limited. We provide a three-step protocol that first involves detergent-based fractionation of the cytoplasmic and nuclear compartments, maintaining the integrity and activity of proteasome complexes. Second, the protocol employs native gel separation of large multiprotein complexes in the fractions and a fluorescence-based in-gel quantitation of the activity and different proteasome particles. Finally, the protocol involves protein in-gel denaturation and transfer to a PVDF membrane. Western blotting then detects and quantifies the different proteasome particles. Therefore, the protocol allows for sensitive measurements of activity and abundance of individual proteasome particles from different cellular compartments. It has been optimized for motor neurons induced from mouse embryonic stem cells but can be applied to a variety of mammalian cell lines.

Original languageEnglish (US)
Article numbere4822
Issue number18
StatePublished - 2023


  • 20S and 26S proteasomes
  • Activity assay
  • Electrophoresis
  • Induced neurons
  • Native protein complexes fractionation

ASJC Scopus subject areas

  • General Neuroscience
  • General Biochemistry, Genetics and Molecular Biology
  • General Immunology and Microbiology
  • Plant Science


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