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A relatively simple and economical protocol for proteomic analyses of human 20S proteasome: Compatible with both scaled‐up and scaled‐down purifications
Author(s) -
Chen Guoqiang,
Luo Yuanming,
Wang Xinli,
Zhao Zhiyun,
Liu Hui,
Zhang Haijing,
Li Zhili
Publication year - 2009
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200800802
Subject(s) - chromatography , chemistry
The 20S core particle (CP) is composed of 28 subunits arranged in four stacked heptameric rings (α7β7β7α7) 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 3.0×10 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.