Blue Native PAGE and Antibody Gel Shift to Assess Bak and Bax Conformation Change and Oligomerization.
Blue native PAGE (BN-PAGE) uses Coomassie dye rather than denaturing SDS to provide a negative charge to proteins for electrophoresis. As such, it is a useful assay for investigating native supramolecular membrane complexes without the need for cross-linking. As Bak and Bax oligomers form in the mitochondrial outer membrane, and they can be efficiently monitored by BN-PAGE. Furthermore, BN-PAGE performed in conjunction with gel-shift using conformation-specific antibodies can provide additional information regarding the activation state of Bak or Bax in specific membrane complexes.
Cell Signalling and Cell Death Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Victoria 3052, Australia;Department of Medical Biology, The University of Melbourne, Melbourne, Victoria 3010, Australia.
Grant
G
Dewson
D000906:Antibodies / Q000032:analysis*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D051336:Mitochondrial Membranes / Q000378:metabolism; D063347:Native Polyacrylamide Gel Electrophoresis / Q000379:methods*; D011487:Protein Conformation; D055503:Protein Multimerization; D013194:Staining and Labeling / Q000379:methods*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein