title: Characterization of the proteasome using native gel electrophoresis.
abstract: Several features of the proteasome make it an excellent subject for analysis by native gel electrophoresis: its size, the multiplicity of variant complexes having proteasome activity, the ease of in-gel assays for proteasome activity, and even its relatively high cellular abundance. Accordingly, native gels have been used to analyze the composition, assembly, gating activity, and binding characteristics of the proteasome. This chapter describes methods for preparing, running, and developing native gels and the proteasome species that are routinely visualized. Additionally, the use of native gels to resolve proteasome complexes present in lysate and to characterize proteasome ligands are described. Following native gel electrophoresis, secondary analyses can be performed, such as activating the core particle, making specific activity assessments, Western blotting of the native gel, resolving native complexes with subsequent SDS-PAGE, and protein identification by mass spectrometry.
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Elsasser
authors.forename: Marion
authors.initials: M
authors.lastname: Schmidt
authors.forename: Daniel
authors.initials: D
authors.lastname: Finley
mesh_terms: D000818:Animals; D015153:Blotting, Western / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism; D011817:Rabbits; D012394:Rosaniline Dyes; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D016622:Silver Staining / Q000379:methods; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D012394:Rosaniline Dyes; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C048139:Coomassie blue; D046988:Proteasome Endopeptidase Complex