Cytochrome c Releasing Apoptosis Assay Kit (ab65311) provides an effective means for detecting cytochrome c translocation from mitochondria into cytosol during apoptosis. The kit provides unique formulations of reagents to isolate a highly enriched mitochondria fraction from cytosol. The procedure is so simple and easy to perform; no ultracentrifugation is required and no toxic chemicals are involved. Cytochrome c releasing from mitochondria into cytosol is then determined by Western blotting using the cytochrome c antibody provided in the kit. Visit our FAQs page for tips and troubleshooting.
The anti-Cytochrome c antibody is a mouse monoclonal antibody that reacts with denatured human, mouse, and rat cytochrome c.
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.
|5X Cytosol Extraction Buffer||1 x 20ml|
|Anti-Cytochrome c Mouse mAb||Green||1 x 500µl|
|DTT||1 x 110µl|
|Mitochondria Extraction Buffer||1 x 10ml|
|Protease Inhibitor Cocktail||1 vial|
Our Abpromise guarantee covers the use of ab65311 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Predicted molecular weight: 11.7 kDa.|
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.