Description
Key facts
Sample types:Tissue, Suspension cells, Adherent cells,
Reacts with:Mouse, Rat, Human,
Assay type:Direct,
Assay time:3h
Product details:
Cytochrome c Release Assay Kit ab65311 provides an effective means for detecting cytochrome c translocation from mitochondria into cytosol during apoptosis.
The kit provides reagents to isolate a highly enriched mitochondria fraction from cytosol. The procedure is simple and easy to perform; no ultracentrifugation is required and no toxic chemicals are involved.
Cytochrome c release from mitochondria into the cytosol is determined by Western blotting using the cytochrome c antibody provided in the kit.
The anti-Cytochrome c antibody is a mouse monoclonal antibody that reacts with denatured human, mouse, and rat cytochrome c.
Cytochrome c release assay protocol summary:
- collect cells, centrifuge and wash with PBS
- resuspend in cytosol extraction buffer mix
- homogenize cells with a dounce tissue grinder
- centrifuge homogenate at 700 x g for 10 min
- collect supernatant and centrifuge at 10,000 g for 30 min, collect supernatant as cytosolic fraction
- resuspend pellet in mitochondrial extraction buffer mix and save as mitochondrial fraction
- analyze cytosolic and mitochondrial fractions in western blotting with cytochrome c antibody
This kit was previously called Cytochrome c Releasing Apoptosis Assay Kit. Cytochrome c plays an important role in apoptosis. The protein is located in the space between the inner and outer mitochondrial membranes. An apoptotic stimulus triggers the release of cytochrome c from the mitochondria into cytosol where it binds to Apaf-1. The cytochrome c/Apaf-1 complex activates caspase-9, which then activates caspase-3 and other downstream caspases. **Other apoptosis assays** For more apoptosis assays, review the .
The Safety Datasheet for this product has been updated for certain countries. Please check the current version in the Support and downloads section.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information--20°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Cytochrome c also known as cyt c is a small heme protein with a mass of approximately 12 kDa. It is expressed in the mitochondria of eukaryotic cells across different species. Its mechanical role involves electron transfer within the mitochondrial electron transport chain. Cytochrome c transfers electrons between complex III (cytochrome bc1 complex) and complex IV (cytochrome c oxidase complex) playing a critical role in cellular respiration and energy production.
Biological function summary
Cytochrome c participates in both energy generation and apoptosis. It functions as part of the electron transport chain complex contributing to the maintenance of the mitochondrial membrane potential. When released into the cytosol cytochrome c initiates the apoptotic cascade by binding to Apaf-1 which then forms the apoptosome complex along with procaspase-9. This triggers subsequent caspase activation leading to programmed cell death.
Pathways
Cytochrome c plays a central role in the intrinsic pathway of apoptosis and oxidative phosphorylation. In the apoptotic pathway after release from mitochondria it interacts with Apaf-1 and procaspase-9 eventually activating caspase-3 and other executioner caspases. Additionally during oxidative phosphorylation cytochrome c works closely with cytochrome bc1 and cytochrome c oxidase in the electron transport chain facilitating ATP synthesis.
Cytochrome c is linked with conditions such as cancer and neurodegenerative diseases. Its release from mitochondria is associated with apoptotic cell death which can either suppress tumor development or contribute to cancer cell resistance. In neurodegenerative disorders like Alzheimer's disease impaired cytochrome c function can result in cellular energy deficits and increased apoptosis. The interaction with Apaf-1 is vital in these processes highlighting the importance of cytochrome c in pathology.
