Detection of PARP-induced ADP-ribosylation using a BMG LABTECH microplate reader
Inhibition of the DNA repair proteins Poly [ADP-ribose] polymerases (PARP) became a promising option for treatment of cancers mutated in other DNA repair factors. Those mutations are found in subtypes of breast or ovarian cancer. For marking sites of DNA damage, PARP transfers ADP onto itself or onto the surrounding chromatin and extends it to Poly-ADP-ribose chains by using NAD+ as a substrate.
In order to measure PARP activity, the enzyme is firstly immobilized on a microplate and biotinylated NAD+ substrate is added. Biotinylated ADP that was transferred by PARP is then detected by binding of a streptavidin-coupled HRP, subsequent substrate addition and acquisition of chemiluminescence.
Reading the assay on CLARIOstar® gives a linear signal for a wide range of enzyme concentrations and allows for determination of the kinetic parameter Km. Further, IC50 of the clinically relevant PARP inhibitor Olaparib could be calculated.