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.
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.
|Product Includes||Volume||Solution Color|
|Cleaved PARP (Asp214) Rabbit mAb Coated Microwells||96 tests|
|PARP Rabbit Detection mAb (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|
|Sealing Tape||2 ea|
|ELISA Wash Buffer (20X) 9801||25 ml|
|ELISA Sample Diluent||25 ml||Blue|
|Cell Lysis Buffer (10X) 9803||15 ml|
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.
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 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.
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).
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