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| A method for purification of 20S proteasome (20S core particle, CP) was developed by combining differential centrifugations with nondenaturing polyacrylamide gel electrophoresis (native-PAGE), irrespective of species origins of CPs. CP purified from human erythrocytes was subjected to proteomic analysis by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), revealing 33 spots of subunit isoforms with different molecular weights and isoelectric points, more than 14 constituent subunits. Furthermore, other four CPs were purified from yeast, mouse liver, two pancreatic cancer cell lines SW1990 and PANC-1 using this method mentioned above, and subjected to proteasome heterogeneity analysis by two-dimensional native/SDS-PAGE (native/sodium dodecyl sulphate polyacrylamide gel electrophoresis), together with CP from erythrocytes. In conclusion, the method described acts as a rapid and effective tool for CP isolations, and the results obtained may be served as a footstone for investigations of the species-dependent proteasome heterogeneity |
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| Keywords:Proteomics; Proteasome; Heterogeneity; Nondenaturing polyacrylamide gel electrophoresis; Differential centrifugation; SW1990 cell line |
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