ApoAlert Cell Fractionation Kit
The ApoAlert Cell Fractionation Kit provides an effective way to isolate a highly enriched mitochondrial fraction from the cytoplasm of apoptotic and nonapoptotic cells. With this kit, you can easily determine if cytochrome c has been released from the mitochondria and is present in the cytosolic fraction―an indicator of mitochondrial involvement in apoptosis. The procedure is fast and simple, with no ultracentrifugation required.

Cytochrome c has been shown to play a major role in apoptosis (1, 2). This soluble protein is localized in the space between the inner and outer mitochondrial membranes. An apoptotic stimulus triggers the release of cytochrome c from the mitochondria into the cytosol, where it initiates the caspase cascade by binding to Apaf-1. The cytochrome c/Apaf-1 complex activates caspase-9, which then activates caspase-3 and other downstream caspases.

The ApoAlert Cell Fractionation Kit allows you to separate a mitochondrial-enriched fraction from the cytosol with just two standard centrifugation steps. The kit also includes two antibodies for visualizing the fractions. The COX4 Antibody detects cytochrome c oxidase subunit IV (COX4), a protein that is localized to the inner mitochondrial membrane and remains in the mitochondria during apoptosis. This antibody allows you to determine the mitochondrion-enriched fraction (Figure 1, Panel A). The second antibody is the Cytochrome c Antibody. With this antibody you can determine which fraction contains cytochrome c by probing Western blots of cytosolic and mitochondrial fractions (Figure 1, Panels B & C). A positive result for the presence of cytochrome c in the cytosolic fraction indicates that cytochrome c was released from the mitochondria.

The ApoAlert Cell Fractionation Kit provides optimized reagents and antibodies for 100 assays.
그림 1. Western blot detection of cytochrome c oxidase subunit IV and cytochrome c. Apoptosis was induced with staurosporine in NIH/3T3 cells. Six hours after induction, the mitochondrial and cytosolic fractions were isolated using the ApoAlert Cell Fractionation Kit. Panel A. Mitochondrial and cytosolic fractions from induced cells; probed with COX4 Antibody. Panel B. Cytosolic fractions from induced and uninduced cells; probed with Cytochrome c Antibody. Panel C. Mitochondrial fractions from induced and uninduced cells; probed with Cytochrome c Antibody.
적용분야
Assay for mitochondrial involvement in apoptosis
References
1. Liu, X., et al. (1996) Cell 86(1):147-157.
2. Peng, L., et al. (1997) Cell 91(4):479-489.
구성물
Cell Fractionation Buffer
Cell Wash Buffer
Protease inhibitor
DTT
Cytochrome c Antibody
COX4 Antibody
User Manual (PT3324-1)
보관조건
4°C for Buffers
-20°C for all other components