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Because cytochrome c plays a major role in apoptosis, its measurement can lead to greater understanding of certain diseases on a cellular level. Cytochrome c is an essential component of the mitochondrial respiratory chain. It is a soluble protein that is localized in the intermembrane space and loosely attached to the surface of the inner mitochondrial membrane. Cytochrome c is important to the process of creating cellular energy, the main function of mitochondria. When mitochondria are damaged, cytochrome c is released into the main body of the cell and, if the cell itself is damaged, into the surrounding tissue. The release of cytochrome c is part of the cascade of cellular events that leads to apoptosis, or programmed cell death. When the cycle that leads to apoptosis begins, cytochrome c appears outside the mitochondria within one hour.
FunctionELISA Cytochrome c uses a Capture Antibody specific for cytochrome c, a biotinylated Detecting Antibody and Horseradish peroxidase-conjugated Streptavidin to provide colorimetric detection that enables accurate study of cytochrome c in response to external stimuli. This FunctionELISA kit requires the use of a microplate reader. For additional tools for the study of cytochrome c, see the Mitochondrial Fractionation Kit.
Figure 1: Location of cytochrome c in HeLa cells.
HeLa cells were grown to 90-95% confluence and treated with 10 µM Actinomycin D. Cells were harvested at 0 hours and after 16 hours of induction and mitochondrial and cytosolic extracts were isolated using the Mitochondrial Fractionation Kit. Two µg of each lysate was tested using the FunctionELISA Cytochrome c Kit (Figure 1A) and Western blot analysis using a 4-20%Tris-Glycine gel (Figure 1B).
Lane 1: Cytosolic extract, no induction
Lane 2: Mitochondrial extract, no induction
Lane 3: Cytosolic extract, 16 hr post-induction
Lane 4: Mitochondrial extract, 16 hr post-induction
Lane 5: Protein standard
