Analyzing lipid raft dynamics during cell apoptosis.
Increasing lines of evidence suggest a role for lipid rafts, glycosphingolipid-enriched microdomains, in cell life and death. This chapter describes in brief the methods used to analyze raft interactions with proteins involved in apoptosis. This chapter focuses mainly on coimmunoprecipitation methods, which represent a useful tool in analyzing raft dynamics during apoptosis. Glycosphingolipid analysis in the immunoprecipitates is performed by thin-layer chromatography. Moreover, methods for the analysis of mitochondrial raft-like microdomains are also described. Detergent (Triton X-100)-insoluble material from isolated mitochondria can be analyzed by Western blot. Further insights can also come from both light and electron microscopy analyses. These can provide useful information as concerning lipid raft distribution at the cell surface or in the cell cytoplasm. Paradigmatic micrographs are shown. The combined use of all these different approaches appears to be mandatory for analyzing the role of lipid raft dynamics during apoptosis.
Malorni
Walter
W
Department of Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy.
Garofalo
Tina
T
Tinari
Antonella
A
Manganelli
Valeria
V
Misasi
Roberta
R
Sorice
Maurizio
M
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D006801:Humans; D047468:Immunoprecipitation; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism
D008563:Membrane Lipids