TR-FRET PARP2 Trapping Assay Kit

The TR-FRET PARP2 Trapping Assay Kit is designed to detect the poly-ADP-ribosylation activity of PARP2 and the status of PARP2 trapping on DNA. The DNA substrate in the kit is labeled with a fluorophore (acceptor). A Terbium (Tb)-labeled anti-Tag2 antibody that binds to Tag2-Kras serves as the fluorescence donor. Activation of Tb results in fluorescence resonance energy transfer (FRET) if PARP2 binds to the fluorescence-labeled DNA, since the binding brings the fluorescence donor into close proximity with the fluorophore acceptor. Thus, the binding status can be quantitatively measured by calculating the ratio of the emission fluorescence intensities of the acceptor (665 nm) and the donor (620 nm). In the presence of NAD⁺, auto-PARylation of PARP2 leads to its dissociation from DNA, resulting in a decrease in the FRET signal. Inhibition of auto-PARylation activity traps PARP2 on the DNA, and the FRET signal remains high.

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Background

PARP2 (Poly (ADP-ribose) polymerase 2) is a member of the PARP family and plays a crucial role in DNA repair, particularly in the repair of single-strand breaks (SSBs) in DNA. It binds to DNA at the site of damage, becomes catalytically activated, and uses NAD⁺ as a substrate to add poly (ADP-ribose) (PAR) chains to itself and other proteins-a process called PARylation that results in the recruitment of other DNA repair proteins to the damaged site. Because of the high negative charge of PAR polymers, extensive autoPARylation of PARP2 leads to the dissociation of PARP2 from DNA, which is required for DNA repair completion. PARP2 is often overexpressed in various cancers, including breast, ovarian, prostate, lung, and glioblastoma. This overexpression is thought to support tumor cell survival. Some PARP inhibitors not only block the catalytic activity of PARP2 but also trap PARP2 on DNA at sites of damage, preventing its release. This creates a toxic DNA-protein complex that interferes with DNA replication and repair, leading to cell death, particularly in cancer cells deficient in homologous recombination repair (e.g., BRCA1/2-mutant cells).