Product Pathways - PathScan ELISA
PathScan® Cleaved Caspase-3 (Asp175) Sandwich ELISA Kit #7190
| Kit Includes | Volume | Solution Color |
|---|---|---|
| Caspase-3 (7190-C3) Ab coated Microwells | ||
| Cleaved Caspase-3 Detection Ab | 11 milliliters | |
| HRP-linked Streptavidin | 11 milliliters | |
| TMB Substrate | 11 milliliters | Colorless |
| STOP Solution | 11 milliliters | Colorless |
| Sealing Tape | 2 sheets | |
| 20X Wash Buffer | 25 milliliters | Colorless |
| Sample Diluent | 25 milliliters | Blue |
| Cell Lysis Buffer (10X) # 9803 | 15 milliliters | Yellowish |
Note: 12 8-well modules –Each module is designed to break apart for 8 tests.
Note: Kit should be stored at 4°C with the exception of Cell Lysis Buffer (10X), which is stored at –20°C (packaged separately).
Species Cross-Reactivity
H
Reactivity Key: H=Human
Description
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 total Caspase-3 Antibody (7190-C3*) has been coated onto the microwells. After incubation with cell lysates, the caspase-3 (cleaved and uncleaved) protein is captured by the coated antibody. Following extensive washing, A biotinylated Cleaved Caspase-3 Antibody (#9661*) 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.
Specificity / Sensitivity
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).
Western Blotting
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).
Sequence
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 37°C for 3 hours.
Background
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 aspartic acid at the P1 position (2).
- Fernandes-Alnemri, T. et al. (1994) J. Biol. Chem. 269, 30761-30764.
- Nicholson, D. W. et al. (1995) Nature 376, 37-43.
- Steiner, P. et al. (2007) Clin Cancer Res 13, 1540-51.
Application References
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