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CST’s PathScan® Apoptosis Multi-Target Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that combines the reagents necessary to detect endogenous levels of p53 protein, phospho-p53 protein (Ser15), Bad, phospho-Bad (Ser112), Cleaved Caspase-3 (Asp175) and Cleaved PARP (Asp214). These molecules represent key signaling proteins in pathways controlling survival and apoptosis. Sixteen assays are provided for each target protein. Specific assay formulations for the indicated target proteins can be found in the datasheets associated with the individual sandwich ELISA kits*. Briefly, a capture antibody** has been coated onto the microwells. After incubation with cell lysates, the target protein is captured by the coated antibody. Following extensive washing, a detection antibody** is added to detect the captured target protein. An HRP-linked secondary antibody is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of absorbance for this developed color is proportional to the quantity of bound target protein.
*See companion products.
**Antibodies in kit are custom formulations specific to kit.
CST's PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105 detects endogenous levels of six proteins: total p53, phospho-p53 (Ser15), total Bad, phospho-Bad (Ser112), cleaved caspase-3 (Asp175) and cleaved PARP (Asp214). Activation of these proteins can be observed over time in response to toxic chemical compounds. As shown in Figures 1 and 2, both doxorubicin and staurosporine can induce apoptosis in HeLa cells, evidenced by increased levels of cleaved PARP and caspase-3. However, treatment with doxorubicin, which damages cellular DNA, induces p53 phosphorylation at Ser15 and stabilizes p53, while treatment with staurosporine, a kinase inhibitor, has no effect on p53 phosphorylation. While total Bad and phospho-Bad (Ser112) levels are relatively consistent after doxorubicin treatment, a gradual decline of both targets was observed after staurosporine treatment. COS cells are resistant to apoptosis due to high constitutive levels of p53. Therefore, the same dose of doxorubicin applied to HeLa cells only induces low amounts of apoptosis in these cells as evidenced by cleaved caspase-3 and cleaved PARP protein levels (Figure 3). The relationship between the protein concentration of the lysate and the absorbance at 450 nm can be found in the datasheets associated with the individual PathScan® Sandwich ELISA Kits*.
*See companion products.
This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Initiator caspases (including 8, 9, 10 and 12) are closely coupled to proapoptotic signals. Once activated, these caspases cleave and activate downstream effector caspases (including 3, 6 and 7), which in turn cleave cytoskeletal and nuclear proteins like PARP, α-fodrin, DFF and lamin A, and induce apoptosis. Cytochrome c released from mitochondria is coupled to the activation of caspase-9, a key initiator caspase (1). Proapoptotic stimuli include the FasL, TNF-α, DNA damage and ER stress. Fas and TNFR activate caspases 8 and 10 (2), DNA damage leads to the activation of caspase-9 and ER stress leads to the calcium-mediated activation of caspase-12 (3). The inhibitor of apoptosis protein (IAP) family includes XIAP and survivin and functions by binding and inhibiting several caspases (4,5). Smac/Diablo, a mitochondrial protein, is released into the cytosol upon mitochondrial stress and competes with caspases for binding of IAPs. The interaction of Smac/Diablo with IAPs relieves the inhibitory effects of the IAPs on caspases (6).
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