title: A relatively simple and economical protocol for proteomic analyses of human 20S proteasome: Compatible with both scaled-up and scaled-down purifications.
abstract: The 20S core particle (CP) is composed of 28 subunits arranged in four stacked heptameric rings (alpha7beta7beta7alpha7) forming a symmetrical barrel-shaped structure. Typically, in previous reports, the purification of CP mainly relied on the antibody, liquid chromatography and affinity tag-based strategies. In this study, we report a relatively simple and economical protocol for proteomic analyses of CP, which combines differential centrifugations with native-PAGE. Furthermore, it is compatible with both scaled-up purification from erythrocytes and scaled-down purification from low to around 3.0 x 10(7) pancreatic cancer cells, SW1990. In addition, a direct three-dimensional gel electrophoresis approach that omits the interval procedure between native-PAGE and IEF/SDS-PAGE is introduced. The results obtained in this study show that this protocol has a valuable potential for the studies of CP and other protein complexes.
authors.affiliation: Department of Biophysics and Structural Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical College, Beijing, P. R. China.
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Chen
authors.forename: Yuanming
authors.initials: Y
authors.lastname: Luo
authors.forename: Xinli
authors.initials: X
authors.lastname: Wang
authors.forename: Zhiyun
authors.initials: Z
authors.lastname: Zhao
authors.forename: Hui
authors.initials: H
authors.lastname: Liu
authors.forename: Haijing
authors.initials: H
authors.lastname: Zhang
authors.forename: Zhili
authors.initials: Z
authors.lastname: Li
mesh_terms: D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry; D006801:Humans; D010190:Pancreatic Neoplasms / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000032:analysis* / Q000097:blood; D040901:Proteomics / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D046988:Proteasome Endopeptidase Complex