title: Purification of large cytosolic proteases for in vitro assays: 20S and 26S proteasomes.
abstract: Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and efficiency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAE-ion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fluorogenic substrates and specific inhibitors.
authors: Institute for Immunology, University of Mainz, Mainz, Germany. tenzer@uni-mainz.de.
authors: Stefan
authors: S
authors: Tenzer
authors: Institute for Immunology, University of Mainz, Mainz, Germany.
authors: Tobias
authors: T
authors: Hain
authors: Institute for Immunology, University of Mainz, Mainz, Germany.
authors: Hendrik
authors: H
authors: Berger
authors: Institute for Immunology, University of Mainz, Mainz, Germany.
authors: Hansjörg
authors: H
authors: Schild
mesh_terms: D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000327:Adsorption; D000645:Ammonium Sulfate / Q000737:chemistry; D015153:Blotting, Western; D005591:Chemical Fractionation / Q000379:methods*; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology*; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays / Q000379:methods*; D004912:Erythrocytes / Q000166:cytology; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification* / Q000378:metabolism*; D061988:Proteasome Inhibitors / Q000494:pharmacology; D059748:Proteolysis; D014461:Ultracentrifugation
chemical_list: D009842:Oligopeptides; D061988:Proteasome Inhibitors; C067713:lactacystin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D005990:Glycerol; D000645:Ammonium Sulfate; D000111:Acetylcysteine; C078846:epoxomicin