Cell Signaling Technology

Product Pathways - PathScan ELISA

PathScan® Cleaved PARP (Asp214) Sandwich ELISA Kit #7262

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Kit Includes Volume Solution Color
Cleaved-PARP (Asp214) Antibody Coated Microwells 96 tests
Biotinylated PARP Detection Rabbit mAb 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
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, which is stored at –20°C (packaged separately).

Species Cross-Reactivity

H M

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

Description

CST's PathScan® Cleaved PARP (Asp214) Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of cleaved PARP (Asp214) protein. A cleaved PARP (Asp214) Rabbit mAb has been coated onto the microwells. After incubation with cell lysates, cleaved PARP (Asp214) protein is captured by the coated antibody. Following extensive washing, a biotinylated PARP Rabbit mAb is added to detect the captured cleaved PARP protein. HRP-linked Streptavidin is then used to recognize the bound detection antibody. HRP substrate, TMB, is added to develop color. The magnitude of the absorbance for this developed color is proportional to the quantity of cleaved PARP (Asp214) protein.Antibodies in kit are custom formulations specific to kit.

Specificity / Sensitivity

CST's PathScan® Cleaved PARP (Asp214) Sandwich ELISA Kit detects endogenous levels of cleaved PARP (Asp214) protein. As shown in Figure 1, using Cleaved PARP (Asp214) ELISA Kit #7262, a significant induction of cleaved PARP (Asp214) is detected in HeLa cells treated with staurosporine. 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

ELISA - Western correlation

Figure 1: Treatment of HeLa cells with staurosporine stimulates cleavage of PARP at Asp214 as detected by PathScan® Cleaved PARP (Asp214) Sandwich ELISA Kit #7262. Absorbance at 450 nm is shown in the top figure, while the corresponding Western blot using Cleaved PARP (Asp214) Antibody #9548 (left panel) or PARP Antibody #9542 (right panel) is shown in the bottom figure. #9542 detects both full-length and cleaved PARP.

Sensitivity

Sensitivity

Figure 2: The relationship between protein concentration of lysates from untreated and staurosporine (STO) treated HeLa cells and the absorbance at 450 nm is shown. HeLa cells (80% confluent) were treated with staurosporine (1 µM) for 3 hours. Absorbance at 450 nm is shown in the top figure, while the corresponding Western blot using Cleaved PARP Antibody #9548 is shown in the bottom figure.

Background

PARP, a 116 kDa nuclear poly (ADP-ribose) polymerase, appears to be involved in DNA repair in response to environmental stress (1). This protein can be cleaved by many ICE-like caspases in vitro (2,3) and is one of the main cleavage targets of caspase-3 in vivo (4,5). In human PARP, the cleavage occurs between Asp214 and Gly215, which separates the PARP amino-terminal DNA binding domain (24 kDa) from the carboxy-terminal catalytic domain (89 kDa) (2,4). PARP helps cells to maintain their viability; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis (6).

  1. Satoh, M.S. and Lindahl, T. (1992) Nature 356, 356-358.
  2. Lazebnik, Y. A. et al. (1994) Nature 371, 346-347.
  3. Cohen, G.M. (1997) Biochem. J. 326, 1-16.
  4. Nicholson, D. W. et al. (1995) Nature 376, 37-43.
  5. Tewari, M. et al. (1995) Cell 81, 801-809.
  6. Oliver, F.J. et al. (1998) J. Biol. Chem. 273, 33533-33539.

Application References

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Protocols

Companion Products

Rabbit Monoclonals are produced under license (granting certain rights, including those under U.S. Patent No. 5,675,063) from Epitomics, Inc.

For Research Use Only. Not For Use In Diagnostic Procedures.

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