title: Isolation and purification of proteasomes from primary cells.
abstract: Proteasomes play an important role in cell homeostasis and in orchestrating the immune response by systematically degrading foreign proteins and misfolded or damaged host cell proteins. We describe a protocol to purify functionally active proteasomes from human CD4(+) T cells and dendritic cells derived from peripheral blood mononuclear cells. The purification is a three-step process involving ion-exchange chromatography, ammonium sulfate precipitation, and sucrose density gradient ultracentrifugation. This method can be easily adapted to purify proteasomes from cell lines or from organs. Methods to characterize and visualize the purified proteasomes are also described.
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland.|Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Steers
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland.|Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland.
authors.forename: Kristina K
authors.initials: KK
authors.lastname: Peachman
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland.
authors.forename: Carl R
authors.initials: CR
authors.lastname: Alving
authors.affiliation: United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland.
authors.forename: Mangala
authors.initials: M
authors.lastname: Rao
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D003713:Dendritic Cells / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D046988:Proteasome Endopeptidase Complex
keywords: antigen processing; immunoproteasomes; proteasomes