Product nameCytochrome c Release Assay Kit
See all Cytochrome c kits
Sample typeTissue, Adherent cells, Suspension cells
Assay time3h 00m
Species reactivityReacts with: Mouse, Rat, Human
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 apoptosis assay and apoptosis marker guide.
Storage instructionsStore at -20°C. Please refer to protocols.
Components Identifier 100 tests 5X Cytosol Extraction Buffer 1 x 20ml Anti-Cytochrome c Mouse mAb Green 1 x 100µl DTT 1 x 110µl Mitochondria Extraction Buffer 1 x 10ml Protease Inhibitor Cocktail 1 vial
- Pathways and Processes
- Metabolic signaling pathways
- Energy transfer pathways
- Integration of energy
RelevanceCytochrome 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.
Cytochrome C release was measured using Cytochrome C releasing apoptosis assay kit (ab65311). Blots showing immunoreactive bands for cytochrome c in cytosol (Image A). Data was expressed in fold-increase of cytochrome c compared to vehicle. Protein expression levels were normalized to β-actin (Figure B). Blots (Image C) of immunoreactive bands for cytochrome C in mitochondria. Figure D shows a fold-increase of cytochrome C compared to vehicle. Protein expression levels were normalized to COX IV.
5x10e7 Jurkat cells were cultured in the absence (1-2) or presence of 2 uM Camptothecin (CPT) (ab120115) for 24 hours (3-4) or with 10 uM CPT for 4 hours (5-6). 30 uL cytosolic (1, 3, 5) and mitochondrial (2, 4, 6) extracts were loaded onto the gel. Membranes were probed with anti-Cytochrome C Mouse MAb (ab65311) (dilution 1:200) followed by Goat Anti-Mouse IgG (HRP) (ab97040) (dilution 1:2000).
Bands were detected at the prediced size of 12 kDa.
This product has been referenced in:
- Kawala RA et al. Kenalog modified by ionizing radiation induces intrinsic apoptosis mediated by elevated levels of reactive oxygen species in melanoma cancer. Oncol Rep 41:1837-1850 (2019). Read more (PubMed: 30569155) »
- Koutsogiannis Z et al. G418 induces programmed cell death in Acanthamoeba through the elevation of intracellular calcium and cytochrome c translocation. Parasitol Res 118:641-651 (2019). Read more (PubMed: 30617503) »