Two-dimensional gel-based analysis of the demolition phase of apoptosis.
Apoptosis is coordinated by members of the caspase family of aspartic acid-specific proteases. To date, over 400 substrates for the apoptosis-associated caspases have been reported and there are likely to be hundreds more yet to be discovered. Global approaches toward identifying proteins cleaved by caspases during apoptosis are now possible and give a more complete perspective on the alterations to the proteome that occur during this complex process. This chapter outlines methods that have been used successfully to visualize the demolition phase of apoptosis by two-dimensional gel electrophoresis coupled with mass spectrometry. It discusses techniques used to generate cell-free extracts from human cells and details how these extracts can be used to activate caspases. The analysis of these extracts by two-dimensional gel electrophoresis is then described, followed by methods used to identify changes to the proteome during the demolition phase of apoptosis.
Lüthi
Alexander U
AU
Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin, Ireland.
Cullen
Sean P
SP
Martin
Seamus J
SJ
D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D002474:Cell-Free System / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*