Figure 1: Treatment of HeLa cells with staurosporine stimulates cleavage of caspase-3 protein, detected by PathScan® Cleaved Caspase-3 (Asp175) Sandwich ELISA Kit #7190 (top figure), but does not affect the level of total caspase-3 protein detected by western blot (bottom figure), using Cleaved Caspase-3 (Asp175) Antibody #9661 (upper panel) or Caspase-3 Antibody #9662 (lower panel).
Figure 2: Linear relationship between protein concentration of staurosporine-treated HeLa cell lysates and kit assay optical density readings. HeLa cells (90% confluence) were treated with staurosporine (1 uM), and lysed after incubation at 37ºC for 3 hours.
|Product Includes||Volume||Solution Color|
|Cleaved Caspase-3 (Asp175) Rabbit mAb Coated Microwells||96 tests|
|Caspase-3 Rabbit Detection Antibody (Biotinylated)||1 ea||Green (Lyophilized)|
|HRP-Linked Streptavidin (ELISA Formulated)||1 ea||Red (Lyophilized)|
|Detection Antibody Diluent||11 ml||Green|
|HRP Diluent||11 ml||Red|
|TMB Substrate 7004||11 ml|
|STOP Solution 7002||11 ml|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Sealing Tape||2 ea|
|Cell Lysis Buffer (10X) 9803||15 ml|
CST's PathScan® Cleaved Caspase-3 (Asp175) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of cleaved caspase-3 protein. A Cleaved Caspase-3 (Asp175) Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, the cleaved caspase-3 protein is captured by the coated antibody. Following extensive washing, a Biotinylated Caspase-3 Rabbit Antibody is added to detect the captured cleaved caspase-3 protein. HRP-linked streptavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of optical density for this developed color is proportional to the quantity of cleaved caspase-3 protein.
Antibodies in kit are custom formulations specific to kit.
NOTE: Prepare solutions with purified water.
*NOTE: Some PathScan® ELISA Kits may include HRP-Linked Streptavidin in place of HRP-Linked Antibody.
NOTE: Initial color of positive reaction is blue, which changes to yellow upon addition of STOP Solution.
posted November 2013
Protocol Id: 204
CST's PathScan® Cleaved Caspase-3 (Asp175) Sandwich ELISA Kit detects endogenous levels of cleaved caspase-3 protein in human cell lines. Using this ELISA kit #7190, a significant induction of cleaved caspase-3 at aspartic acid 175 can be seen in staurosporine-treated HeLa cells, while the level of total caspase-3, analyzed by western blot using Caspase-3 Antibody #9662, remains unchanged (Figure 1). Jurkat cells treated with etoposide show similar results (data not shown). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.
Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is a critical executioner of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins, such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP) (1). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Cleavage of caspase-3 requires the aspartic acid residue at the P1 position (2).
Explore pathways + proteins related to this product.
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's 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's 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.