PARPtrap™ Assay Kit for PARP

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.  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 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. Further understanding of the molecular pathways involving PARP1, and this contribution to disease, will continue to pave the way for new therapies for PARP1-linked diseases.

96 ReactionsCatalog #NameAmountStorage80501PARP1, GST-tag*1 µg-80°C7827325 nM Fluorescent Labeled Oligonucleotide Duplex100 µl-80°C5x PARPtrap™ Assay Buffer2 x 1 ml-80°C10x NAD+500 µl-80°C79685Black 96-well plate1Room Temp* The initial concentration of enzyme is lot-specific and will be indicated on the tube containing the protein.384 ReactionsCatalog #NameAmountStorage80501PARP1, GST-tag*2 x 1 µg-80°C7827325 nM Fluorescent Labeled Oligonucleotide Duplex4 x 100 µl-80°C5x PARPtrap™ Assay Buffer4 x 1 ml-80°C10x 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.