Characterization of the proteasome using native gel electrophoresis.
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.
Elsasser
Suzanne
S
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Schmidt
Marion
M
Finley
Daniel
D
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
D012394:Rosaniline Dyes; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; C048139:Coomassie blue; D046988:Proteasome Endopeptidase Complex