Poly (ADP-ribose) polymerases (PARPs) are a family of enzymes involved in various cellular processes, including DNA repair, genomic stability, and programmed cell death. PARP enzymes detect and respond to DNA damage by adding ADP-ribose polymers to target proteins, facilitating the recruitment of repair proteins to sites of damage. Dysregulation of PARP activity is implicated in a variety of diseases, particularly cancer, where PARP inhibitors have emerged as a promising therapeutic strategy, especially in tumors with defective DNA repair mechanisms, such as BRCA1/2-mutated cancers.

PARP Enzymatic Assays

We use the ELISA method to detect the enzymatic activity of the PARP family of ADP-ribose polymerases. Histones are coated on the plate, and PARP proteins (PARP1/2/3 require single-strand DNA breaks for activation) along with biotin-labeled NAD are added. As the reaction progresses, PARP1 forms biotin-labeled ADP-ribose chains on the histones. Then, streptavidin-labeled HRP and an ultrasensitive detection reagent are added to detect the signal.

PARP Trapping Assay

When PARP forms a complex with DNA and NAD, it dissociates from the DNA as ADP-ribose chains are generated. Biotin labels are added to single-stranded DNA breaks, and PARP proteins are tagged with His tags. When inhibitors are added, PARP does not dissociate from the DNA, allowing for the detection of fluorescence resonance energy transfer signals between donor and acceptor tags.

PARP Binding Assay

We perform binding assay for PARP1 and PARP2 using both Fluorescence Polarization (FP) and Surface Plasmon Resonance (SPR). The FP assay enables rapid, high-throughput assessment of small molecule binding affinity to the target proteins, while SPR allows for real-time, kinetic analysis of binding interactions, providing insights into binding and dissociation rates. Together, these methods form a robust approach for comprehensive profiling of PARP1 and PARP2 binding properties.