PARPtrap™ Combo Assay Kit for PARP1 and PARP2

PARP1, also known as poly- (ADP-ribose) polymerase 1 or NAD+ADP-ribosyltransferase 1, is part of the PARP family, and it is the most abundant member.  ADP ribosylation, which is the addition of an ADP-ribose to a protein, is a reversible post-translational modification of proteins mostly involved in the DNA Damage Response (DDR) pathway. Poly-ADP-ribosylation (termed PARylation) is the addition of linear or branched chains of ADP-ribose. PARP1 participates in DNA repair by non-homologous end joining (NHEJ), homologous recombination (HR), microhomology-mediated end-joining (MMEJ) and nucleotide excision repair. Dysfunction of DDR pathways can lead to oncogenesis. Overexpression of PARP1 has been found in breast and colon cancer, neuroblastoma, and others. This overexpression can lead to increasing MMEJ, an error-prone DNA repair mechanism, and genome instability leading to cancer. In addition to being involved in DDR, PARP1 is also linked to inflammation and type I diabetes.  PARP2 participates in DNA repair (only 10% of the PARP activity is due to PARP2), but also in oxidative stress and mitochondrial fragmentation. Dysfunction of the DDR and oxidative stress pathways can lead to oncogenesis. Genetic ablation of PARP2 has indicated roles of PARP2 in adipogenesis, spermatogenesis and thymocyte survival. It is also a co-factor of nuclear receptors like ER (estrogen receptor) and PPAR (peroxisome proliferator-activated receptors) . PARP2 is overexpressed in prostate cancer (PCa) and may contribute to the disease through the FOXA1 (forkhead box protein A1) /AR pathway. PARP1 and PARP2 are known to bind damaged DNA through its DNA-binding domains. Binding to DNA activates PARP1/2 and in the presence of NAD+PARP1/2 ribosylates itself (auto-ribosylation) and dissociates from the DNA due to the accumulated negative charge of the ribosyl polymer. In the presence of some inhibitors, however, PARP remains bound to DNA, a phenomenon termed trapping. Trapped PARP-DNA complexes have been shown to be highly cytotoxic to cancer cells, making such inhibitors desirable in cancer therapy. Further understanding of the molecular pathways involving PARP1/2, and their contribution to disease, will continue to pave the way for new therapies for PARP1/2-linked diseases.

Screen small molecules that enhance PARP1/2 trapping onto DNA for drug discovery and high throughput screening (HTS) applications, and IC50determination.

Catalog #NameAmountStoragePARP1 specific reagents80501PARP1, GST-Tag*1 µg-80°C7827325 nM Fluorescent Labeled Oligonucleotide Duplex2 x 100 µl-80°C826745x PARPtrap™ Assay Buffer2 x 1 ml-80°CPARP2 specific reagents80502PARP2, GST-Tag*8 µg-80°C78297100 nM Fluorescent Labeled Nicked Oligonucleotide Duplex2 x 12.5 µl-80°C826755x PARPtrap™ Assay Buffer 22 x 1 ml-80°C827350.5 M DTT200 µl-20°C8267610x NAD+2 x 500 µl-80°C79961Black 384-well plate1Room Temp* The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.

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The final concentration of DMSO in the assay should not exceed 1%.Fluorescent compounds that have λex=470 (5 nm bandwidth) and detection at λem=518 (10 nm bandwidth) can interfere with the readouts.