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PathScan® Apoptosis Multi-Target Sandwich ELISA Kit

PathScan^® Apoptosis Multi-Target Sandwich ELISA Kit #7105

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Product Specifications

REACTIVITY

H Mk

Application Key:

ELISA-ELISA

Species Cross-Reactivity Key:

H-Human
Mk-Monkey

Product Information

Product Description

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.

Protocol

Specificity / Sensitivity

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.

Species Reactivity:

Human, Monkey

Background

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 FasL, TNF-α, DNA damage and ER stress. Fas and TNFR activate caspase-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 IAPs on caspases (6).

Alternate Names

ADP-ribosyltransferase (NAD+); ADP-ribosyltransferase diphtheria toxin-like 1; ADP-ribosyltransferase NAD(+); ADPRT; ADPRT 1; ADPRT1; Antigen NY-CO-13; Apopain; ARTD1; BAD; BBC2; BBC6; BCC7; Bcl-2-binding component 6; Bcl-2-like protein 8; BCL-X/BCL-2 binding protein; Bcl-XL/Bcl-2-associated death promoter; Bcl2 antagonist of cell death; BCL2 associated agonist of cell death; BCL2-antagonist of cell death protein; Bcl2-associated agonist of cell death; BCL2-binding component 6; BCL2-binding protein; Bcl2-L-8; BCL2L8; BMFS5; CASP-3; CASP3; caspase 3; caspase 3, apoptosis-related cysteine peptidase; caspase 3, apoptosis-related cysteine protease; Caspase-3; Caspase-3 subunit p12; Caspase-3 subunit p17; caspase3; Cellular tumor antigen p53; CPP-32; CPP32; CPP32B; Cysteine protease CPP32; DNA ADP-ribosyltransferase PARP1; FLJ92943; LFS1; mutant tumor protein 53; NAD(+) ADP-ribosyltransferase 1; P53; p53 antigen; p53 transformation suppressor; p53 tumor suppressor; pADPRT-1; PARP; PARP cleavage protease; PARP-1; PARP1; Phosphoprotein p53; poly (ADP-ribose) polymerase; poly (ADP-ribose) polymerase 1; poly (ADP-ribose) polymerase family, member 1; Poly [ADP-ribose] polymerase 1; poly(ADP-ribose) polymerase; poly(ADP-ribose) polymerase 1; poly(ADP-ribose) synthetase; poly(ADP-ribosyl)transferase; Poly[ADP-ribose] synthase 1; PPOL; procaspase3; Protein poly-ADP-ribosyltransferase PARP1; Protein Yama; SCA-1; SREBP cleavage activity 1; TP53; transformation-related protein 53; TRP53; tumor protein 53; tumor protein p53; Tumor suppressor p53; tumor supressor p53; Yama

Database Links

UniProt ID: P04637 , Q92934 , P42574 , P09874

Entrez-Gene Id: 7157 , 572 , 836 , 142

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not for Use in Diagnostic Procedures.

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PathScan is a registered trademark of Cell Signaling Technology, Inc.

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PathScan® Apoptosis Multi-Target Sandwich ELISA Kit #7105