Scalable In Vitro Proteasome Activity Assay

Methods Mol Biol. 2018;1844:321-341. doi: 10.1007/978-1-4939-8706-1_21.

Abstract

We developed a degradation assay based on fluorescent protein substrates that are efficiently recognized, unfolded, translocated, and hydrolyzed by the proteasome. The substrates consist of three components: a proteasome-binding tag, a folded domain, and an initiation region. All the components of the model substrate can be changed to modulate degradation, and the assay can be performed in parallel in 384-well plates. These properties allow the assay to be used to explore a wide range of experimental conditions and to screen proteasome modulators.

Keywords: High-throughput degradation assay; Proteasome; Ubiquitin-proteasome system.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biological Assay* / methods
  • Chromatography, Affinity
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Models, Molecular
  • Proteasome Endopeptidase Complex / chemistry
  • Proteasome Endopeptidase Complex / isolation & purification
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Binding
  • Protein Conformation
  • Proteolysis
  • Substrate Specificity
  • Ubiquitin / metabolism

Substances

  • Ubiquitin
  • Proteasome Endopeptidase Complex