A relatively simple and economical protocol for proteomic analyses of human 20S proteasome: Compatible with both scaled-up and scaled-down purifications.
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.
Chen
Guoqiang
G
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.
Luo
Yuanming
Y
Wang
Xinli
X
Zhao
Zhiyun
Z
Liu
Hui
H
Zhang
Haijing
H
Li
Zhili
Z
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
D046988:Proteasome Endopeptidase Complex