PARPtrap™ Combo Assay Kit for PARP1 and PARP2
The PARPtrap™ Combo Assay Kit for PARP1 and PARP2 is a kit designed to measure DNA complex formations of PARP1 and PARP2 enzymes in a high throughput screening assay using fluorescence polarization (FP). PARP1 and PARP2 enzymes are known to bind damaged DNA through their DNA-binding domains. Binding to DNA activates these enzymes and in the presence of NAD+, PARP1 and PARP2 ribosylate themself (autoribosylation), leading to their dissociation from the DNA due to the accumulated negative charge of the ribosyl polymer. In the presence of some inhibitors, however, these PARPs remain bound to the DNA, a phenomenon termed trapping. Trapped PARP-DNA complexes have been shown to be highly cytotoxic to cancer cells, therefore such inhibitors may be
desirable for cancer treatment.
The key to the PARPtrap™ Combo Assay Kit are the fluorescentlabeled DNA probes recognized by PARP1 and PARP2. In the absence of ribosylation, PARP1/2 binds to the DNA fluorescent probe, forming a large complex and resulting in the emission of highly polarized light. However, after autoribosylation, PARP1/2 dissociates from the oligonucleotide duplex, which then rotates freely, emitting less polarized light (Fig. 1). Addition of a PARP inhibitor results in trapping of PARP1/2 to the fluorescent-labeled DNA, and increases the FP signal in a dose dependent manner.
The combo kit is particularly useful to directly and specifically compare, in a single assay, the effect and potency of a compound on PARP1 and PARP2.
The PARPtrap™ Combo Assay Kit for PARP1 and PARP2 is a homogeneous fluorescence polarization assay. The FP signal is measured using a fluorescent microplate reader capable of measuring fluorescence polarization.
Figure 1. PARPtrap™ Combo Assay Kit for PARP1 and PARP2 schematic
Need us to run inhibitor screens or profile your compounds against PARPtrap™ for PARP1 and PARP2? Check out our PARP/PARPTrap™ Screening Services or DNA Replication and Repair Services.
- DTT
- Fluorescent microplate reader capable of measuring fluorescence polarization
- Adjustable micropipettor and sterile tips
- Rotating or rocker platform
| Catalog # | Name | Amount | Storage | |
| PARP1 specific reagents | 80501 | PARP1, GST-tag* | 1 µg | -80°C |
| 78273 | Fluorescent labeled oligonucleotide duplex (25 nM)** | 100 µl | -80°C | |
| 5x PARPtrap™ assay buffer | 2 x 1 ml | -80°C | ||
| PARP2 specific reagents | 80502 | PARP2, GST-tag* | 8 µg | -80°C |
| 78297 | Fluorescent labeled nicked oligonucleotide duplex (100 nM)** | 12.5 µl | -80°C | |
| 5x PARPtrap™ assay buffer 2 | 2 x 1 ml | -80°C | ||
| 10x NAD+ | 2 x 500 µl | -80°C | ||
| 79961 | Black 384-well plate | 1 | Room Temp |
*The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.
**Note that each PARP assay protocol uses its own fluorescent DNA substrate
1. Murai, J. et al. Molecular Cancer Therapeutics 2014. 13: 433-443
2. Murai, J. al. Cancer Research 2012. 72: 5588-5599
3. Zandarashvili, L. et al. Science 2020. 368(6486): 30-31
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