Cytochrome c Release Assay Kit (ab65311)
Key features and details
- Assay type: Direct
- Assay time: 3 hr
- Sample type: Adherent cells, Suspension cells, Tissue
Overview
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Product name
Cytochrome c Release Assay Kit -
Sample type
Tissue, Adherent cells, Suspension cells -
Assay type
Direct -
Assay time
3h 00m -
Species reactivity
Reacts with: Mouse, Rat, Human -
Product overview
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 -
Notes
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.
Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.
Properties
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Storage instructions
Store 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 -
Research areas
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Relevance
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. -
Cellular localization
Mitochondrial -
Database links
- Entrez Gene: 54205 Human
- Omim: 123970 Human
- SwissProt: P99999 Human
- Unigene: 437060 Human
Images
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Inhibition of cytochrome c release from mitochondria in SK-N-SH cells Sun Q., PLoS One 9(6), Fig 4. doi: 10.1371/journal.pone.0098866. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/
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.
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Functional assays: Cytochrome c Releasing Apoptosis Assay Kit (ab65311)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.