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Cytochrome c is located between the inner and the outer mitochondrial membranes. Once activated by the apoptosis stimulus, cytochrome c is released from mitochondria into cytosol where it couples to Apaf-1. The cytochrome c/Apaf-1 complex further activates the downstream caspases. The Cytochrome c Releasing Apoptosis Assay Kit gives an efficient way for sensing cytochrome c releasing from mitochondria into cytosol under apoptosis. The kit utilizes a special formulation of reagents to isolate enriched mitochondrial fraction from cytosol. The protocol is simple to perform without unltracentrifugations and toxic chemicals. The cytochrome c antibody provided in the kit is then used to determine the Cytochrome c releasing from mitochondria into cytosol by Western blotting.