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Assaying cytochrome C translocation during apoptosis
Nigel J Waterhouse1, Rohan Steel, Ruth Kluck
1Cancer Immunology, Research Division, Peter MacCallum Cancer Center, Melbourne, Australia.
Methods in Molecular Biology (Clifton, N.J.)
|June 3, 2004
Summary
Measuring cytochrome c release from mitochondria to the cytoplasm is key to understanding apoptosis. This chapter details a simple, reproducible digitonin-based cell fractionation method for assaying this crucial protein translocation event.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Apoptosis involves programmed cell death.
- Protein translocation from the mitochondrial intermembrane space to the cytoplasm is a critical step in apoptosis.
- Accurate measurement of this translocation is essential for studying apoptotic pathways.
Purpose of the Study:
- To present a simple and reproducible method for assaying cytochrome c release during apoptosis.
- To highlight the separation of cytosolic and mitochondrial fractions using digitonin.
Main Methods:
- Utilized digitonin for selective permeabilization of the plasma membrane.
- Separated cellular components into cytosolic and mitochondrial fractions.
- Quantified cytochrome c release into the cytosolic fraction.
Main Results:
- The digitonin-based fractionation method proved to be simple and highly reproducible.
- Successfully measured cytochrome c release, indicating apoptotic signaling.
Conclusions:
- Digitonin-based cell fractionation is an effective and reliable technique for studying apoptosis.
- This method facilitates the investigation of mitochondrial protein translocation in apoptotic processes.