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.