title: Affinity purification of mammalian 26S proteasomes using an ubiquitin-like domain.
abstract: The standard methods for the isolation of the 26S proteasomes from mammalian tissues have long involved multiple chromatographic steps. This process led to loss of loosely associated regulatory proteins or cofactors and yielded particles with low functional capacity. Here, we describe a single-step affinity purification of 26S proteasome complexes that preserves the association with many 26S proteasome-interacting proteins. Our approach uses the ubiquitin-like domain of human RAD23B as an affinity bait, which allows the rapid and gentle isolation of 26S proteasomes with high purity. This strategy does not require the genetic introduction of tagged subunits nor expensive antibodies, and therefore can be used to isolate 26S proteasomes from any mammalian tissue or yeast. This method, therefore, is an important new tool to study 26S proteasome function in various models of human diseases that are linked to changes in the ubiquitin proteasome system, for example the increased proteasomal proteolysis seen in muscle wasting or the decreased proteasomal capacity that has been reported in various neurodegenerative diseases.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA, USA.
authors.forename: Henrike C
authors.initials: HC
authors.lastname: Besche
authors.forename: Alfred L
authors.initials: AL
authors.lastname: Goldberg
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D018482:Muscle, Skeletal / Q000166:cytology; D046988:Proteasome Endopeptidase Complex / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C513287:RAD23B protein, human; D014452:Ubiquitins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D045643:DNA Repair Enzymes