Immunodetecting members of the Bcl-2 family of proteins.
The Bcl-2 family of proteins centrally regulates the cellular commitment to apoptosis (programmed cell death). Apoptosis, in turn, is critical for the development and homeostasis of multicellular organisms, and defects in apoptosis contribute to a broad range of human diseases and disorders. Consequently, studying the regulation of Bcl-2 and its homologs has significant implications for biomedical research. The mechanisms by which Bcl-2 family members regulate apoptosis are complex, and include variations in intracellular protein levels, direct protein-protein interactions, conformational changes, and subcellular redistribution. Therefore, all of these factors should be considered when assessing how one, or a group, of these proteins contributes to a cell's commitment to apoptosis. The methods described in this chapter will allow the researcher to gain a greater understanding of how intracellular levels of multiple members of the Bcl-2 protein family can be analyzed simultaneously by immunoblotting and how protein-protein interactions can be studied using immunoprecipitation techniques. In addition, the methodology describes how conformational changes can be observed by immunofluorescence and immunoprecipitation, and how subcellular redistribution of Bcl-2 homologs can be followed.
Lock
Richard B
RB
Children's Cancer Institute Australia for Medical Research, South Australia, Sydney.
Murphy
Kathleen M
KM
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D051023:BH3 Interacting Domain Death Agonist Protein; D000072224:Bcl-2-Like Protein 11; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D003584:Cytological Techniques; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D047468:Immunoprecipitation / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry* / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
D051017:Apoptosis Regulatory Proteins; C494772:BAK1 protein, human; C494811:BCL2L1 protein, human; C000606775:BCL2L11 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D000072224:Bcl-2-Like Protein 11; D002352:Carrier Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein