PARP Assays

Poly(ADP-ribose) polymerase (PARP) proteins play a crucial role in DNA repair mechanisms within cells. These enzymes are involved in the repair of single-strand DNA breaks through the base excision repair pathway. Inhibition of PARP proteins has emerged as a promising strategy in cancer therapy, particularly in tumors with defective DNA repair mechanisms, such as those with BRCA mutations. When PARP is inhibited, it prevents the repair of single-strand breaks, leading to the accumulation of double-strand breaks during DNA replication. In cells with deficient homologous recombination, often seen in certain cancer types, these double-strand breaks become more lethal. Exploiting this vulnerability has led to the development of PARP inhibitors, which selectively target cancer cells with impaired DNA repair pathways. The continued development of new PARP inhibitors with improved selectively and reduced side effects is critical for effective precision medicine in oncology.

Enzymatic Activity by ELISA

ELISA: Histone proteins are coated on a plate. Biotin-labeled NAD+ is added with the PARP enzyme. PARP-mediated ribosylation activity is detected by adding Streptavidin-HRP (horseradish peroxidase), which binds to the newly formed ribosylation branch, and a chemiluminescent or colorimetric HRP substrate.

PARP Trapping

Fluorescence Polarization Assay: The assay uses fluorescent DNA probes that emit differently polarized light depending on PARP binding. Addition of a PARP inhibitor prevents ribosylation and results in the trapping of PARP onto the fluorescent oligonucleotide duplex, which increases the Fluorescence Polarization signal in a dose dependent manner. This is a homogeneous assay.