Cleaved PARP (Asp214) Cellular Assay Kit

Cleaved PARP assay as a relevant marker of cell apoptosis

Cisbio's cell-based homogeneous HTRF® kit for cleaved PARP (Asp214) enables the specific and quantitative detection of endogenous levels of the human, mouse and monkey large fragment (89 kDa) of PARP-1. With its central position downstream from caspases 3 and 7, both activated by extrinsic and intrinsic apoptosis pathways, cleaved PARP-1 represents a pertinent marker of cells undergoing apoptosis. It also comes as a useful complement to the HTRF phospho-BAD assay, used to monitor the activation of the Bcl-2 pro-apoptotic proteins of the intrinsic pathway. Dysregulated apoptosis plays a role in various diseases involving insufficient cell death such as cancer, autoimmunity and persistent infections, whereas excessive apoptosis can contribute to neurodegeneration, and ischaemia. The kit's simple add-and-read protocol eliminates all wash steps for a faster analysis and easy miniaturization, while maintaining a high-quality specific output and offering enhanced convenience over ELISA or WB.

Assay principle

HTRF® - the homogeneous cell-based sandwich immunoassay

Cisbio's cleaved-PARP assay is based on a TR-FRET sandwich immunoassay format using two specific anti-PARP-1 p85 fragment monoclonal antibodies, one labeled with Eu3+ cryptate (donor) and the other with d2 (acceptor). Donor-acceptor proximity leads to a fluorescent TR-FRET signal proportional to the number of cells undergoing apoptosis. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.

After cell lysis, the cleaved PARP-1 p85 fragment can be semi-quantitatively detected using the HTRF cleaved-PARP-Asp214 assay kit reagents and most TR-FRET multimode plate readers.

A simpler, more flexible assay protocol - adapted to your applications

Two-plate assay protocol

For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.

Product performance

1- Cleaved PARP-1 Asp214 detection on human HeLa cells treated with Staurosporine

50,000 human cervical cancer HeLa cells were plated in 96-well plates and incubated for 24h at 37 °C - 5% CO2. After incubating with increasing concentrations of Staurosporine for 4 hours, the medium was removed and cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF Cleaved PARP Asp214 detection reagents were added. The HTRF signal was recorded after a 2h incubation time.

2- Cleaved PARP-1 Asp214 detection on mouse C2C12 cells treated with Staurosporine

50,000 mouse myoblast cells were plated in 96-well plates and incubated for 24h at 37 °C - 5% CO2. After incubating with increasing concentrations of Staurosporine for 4 hours, the medium was removed and cells were lysed with 50 µL of lysis buffer for 30 min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF Cleaved PARP Asp214 detection reagents were added. The HTRF signal was recorded after a 2h incubation time.

3- HTRF assay compared to Western Blot

Jurkat cells were grown in a T175 flask at 37 °C, 5% CO2, and then stimulated for 4 hours with staurosporine. After centrifugation and medium removal, pelleted cells were lysed with 3 mL of supplemented lysis buffer for 30 min at room temperature. Soluble fractions were then collected after a 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by Western Blot and by HTRF.

Using the HTRF cleaved PARP Asp214 cellular assay, just 3,125 cells were sufficient for minimal signal detection whereas 12,500 cells were needed for a Western Blot signal. The HTRF cellular assay displays a better sensitivity than Western Blot.

Simplified pathway

Two pathways activate the effector pro-caspase 3 that cleaved PARP on Asp214, leading to apoptosis.

  • An extrinsic pathway (activated by death ligands like TNF-α, FasL and ApoL that bind to their death receptors), activating the initiator procaspase-8
  • An intrinsic pathway, or mitochondrial pathway (activated by cellular damage like stress, radiation, toxin, hypoxia and growth factor withdrawal), also activating pro-apoptotic factors like BAD, then the initiator procaspase-9

In response to death ligands or cell damage and activation of extrinsic or intrinsic apoptosis pathways, the nuclear enzyme PARP-1 (116 kDa) involved in the repair of damaged DNA is cleaved between Asp214 and Gly215 by activated caspases 3 and 7. The cleavage deactivates the enzyme by separating its N-terminal DNA binding domain p25 (24kDa) from its C-terminal catalytic domain p85 (89kDa), leading to DNA fragmentation and cell apoptosis. Cleaved PARP-1 is considered as an essential marker of apoptosis.

The PARP family comprises 18 members with different structures and functions. Among them, PARP (Poly (ADP-Ribose) Polymerase) is a nuclear enzyme responsible for poly ADP-ribose synthesis, and is essential for the maintenance of genomic integrity. It helps cells to maintain their viability.

The HTRF Cleaved PARP assay is specific for the large p85 fragment of PARP-1 and does not measure the full-length protein. It comes as a useful complement to the HTRF phospho-BAD assay, used as a readout for the intrinsic pathway activation.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
PARP cleaved-Asp214 kit - 500 tests64PARPEG
PARP cleaved-Asp214 kit - 10,000 tests64PARPEH

Companion products

DescriptionCat. noProduct insertMSDS
PARP cleaved-Asp214 kit control lysate64PARTDA
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