Characterizing Bcl-2 Family Protein Conformation and Oligomerization Using Cross-Linking and Antibody Gel-Shift in Conjunction with Native PAGE.
The Bcl-2 family of proteins tightly controls the intrinsic or mitochondrial pathway of apoptosis. This family is subdivided based on function into pro-survival proteins (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, Bfl-1/A1) and pro-apoptotic proteins. The pro-apoptotic subset is further divided into those proteins that initiate the pathway, the BH3-only proteins (including Bim, Puma, Noxa, and Bid), and those that execute the pathway, Bak and Bax. Whether a cell lives or dies in response to apoptotic stress is determined by the interactions of the Bcl-2 family, which is in turn influenced by their conformation. We describe here a protocol to interrogate the interactions and conformation of the Bcl-2 family of proteins under native conditions.
Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia. dewson@wehi.edu.au.|Department of Medical Biology, University of Melbourne, Melbourne, VIC, 3010, Australia. dewson@wehi.edu.au.
Grant
G
Dewson
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000378:metabolism*; D002478:Cells, Cultured; D003432:Cross-Linking Reagents / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D063347:Native Polyacrylamide Gel Electrophoresis / Q000379:methods*; D011487:Protein Conformation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D003432:Cross-Linking Reagents; D019253:Proto-Oncogene Proteins c-bcl-2
Apoptosis; Bak; Bcl-2; Cross-linking; Mitochondria; Native polyacrylamide gel electrophoresis; Oligomerization