Cell Signaling Technology

Product Pathways - PathScan ELISA

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

Kit Includes Volume Solution Color
PARP (Asp 214) Ab coated microwells 96 tests
PARP Detection Antibody 11 milliliters Green
Anti-rabbit IgG HRP-Linked Ab 11 milliliters Red
TMB Substrate 11 milliliters Colorless
STOP Solution 11 milliliters Colorless
Sealing Tape 11 milliliters
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 M

Reactivity Key:  H=Human  M=Mouse

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) Mouse 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, PARP Rabbit mAb* is added to detect the captured cleaved PARP protein. Anti-rabbit IgG, HRP-linked Antibody #7074* 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.

Sandwich ELISA

Sandwich ELISA

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.

Sandwich ELISA

Sandwich ELISA

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

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

Product Pathways

Drug Discovery Tools

Featured Technologies

Protein Classes