Product Pathways - PathScan ELISA

PathScan® Cleaved Caspase-3 (Asp175) Sandwich ELISA Kit #7190

PhosphoSitePlus^® protein, site, and accession data: Casp3

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Kit Includes	Volume	Solution Color
Cleaved-Caspase 3 (Asp175) Rabbit mAb coated Microwells	96 tests
Biotinylated Caspase-3 Rabbit Detection Antibody	11 ml	Green
HRP-linked Streptavidin	11 ml	Red
TMB Substrate #7004	11 ml	Colorless
STOP Solution #7002	11 ml	Colorless
Sealing Tape	2 sheets
ELISA Wash Buffer (20X)	25 ml	Colorless
ELISA Sample Diluent	25 ml	Blue
Cell Lysis Buffer (10X) #9803	15 ml	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

Reactivity Key: H=Human
Species enclosed in parentheses are predicted to react based on 100% sequence homology.

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 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.

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). This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect homologous proteins from other species.

ELISA - Western correlation

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).

Sensitivity

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.

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).

Application References

Levy, D.S. et al. (2009) Blood 113, 1723-9. Applications: ELISA

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Protocols

7190:: Sandwich ELISA

Companion Products

This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.