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Application: Great for screening small molecules that enhance PARP1/DNA trapping for drug discovery and HTS applications.
Background: PARP1 is known to bind damaged DNA through its N-terminal zinc finger domain. After PARP1 ribosylates itself (autoribosylation), it dissociates from DNA due to the accumulated negative charge of the ribosyl polymer. Trapped PARP-DNA complexes have been shown to be cytotoxic to cancer cells.
Description: The PARPtrap™ Assay Kit for PARP1 is designed to measure PARP1/DNA complex formation in a high throughput screening assay using fluorescence polarization (FP). PARP1 is known to bind damaged DNA through its DNA-binding domains. Binding to DNA activates PARP1 and in the presence of NAD+ PARP1 ribosylates itself (auto-ribosylation), what in consequence leads to PARP1 dissociation from the DNA due to the accumulated negative charge of the ribosyl polymer. In the presence of some inhibitors, however, PARP remains 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™ Assay Kit for PARP1 is the fluorescent-labeled oligonucleotide duplex. In the absence of ribosylation, PARP1 binds to the fluorescent probe, forming a large complex and resulting in the emission of highly polarized light. However, after auto-ribosylation, PARP1 dissociates from the oligonucleotide duplex, which then rotates freely, emitting less polarized light (Fig. 1). Addition of a PARP1 inhibitor results in trapping of PARP1 to the fluorescent oligonucleotide duplex, and increases the FP signal in a dose dependent manner.The PARPtrap™ Assay Kit for PARP1 is a fluorescence polarization homogeneous assay. The FP signal is measured using a fluorescent microplate reader capable of measuring fluorescence polarization.
Storage Stability: Up to 1 year when stored as recommended.
Supplied As: The PARPtrap™ Assay Kit for PARP1 enzyme comes in a convenient 96-well format, with purified PARP1 enzyme, fluorescent-labeled nicked oligonucleotide duplex, and PARPtrap™ assay buffer 1 for 100 enzyme reactions.
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
References: 1. Murai, J., et al. Molecular Cancer Therapeutics 2014. 13:433-443.
2. Murai, J., et al. Cancer Research 2012. 72:5588-5599.
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