Analyzing PARP1 Activity: Small Molecule Reactants and Attached Chains of Poly (ADP-Ribose)

We describe a method for analyzing multiple products of PARylation by PARP1 and/or PARP2 using high-pressure liquid chromatography. The method quantitates the small molecules NAD⁺ (the substrate), nicotinamide (the byproduct of PARylation or hydrolysis of NAD⁺), and ADPR, the product of NAD⁺ hydrolysis. The method also quantitates the products of PARylation following digestion of the PAR chains into "ends," "middles," and "branches." This method is useful for dissecting both the activity and the partitioning of PARylation products between different outcomes (i.e., long chains vs. short chains, PARylation vs. hydrolysis).