Quantification of Poly(ADP-Ribose) In Vitro: Determination of the ADP-Ribose Chain Length and Branch

The structural integrity of eukaryotic genomes, to a great extent, depends on highly regulated and �coordinated enzymatic chromosomal poly(ADP-ribosyl)ation cycles that target chromatin proteins for specific covalent epigenetic poly(ADP-ribose) modification. As a result, the accurate determination of poly(ADP-ribosyl)ation amino acid specificity, as well as, a detailed characterization of the structural �complexity of the protein-bound ADP-ribose polymers generated, e.g., linear versus branched ADP-ribose chains, need to be carefully sorted out. In this chapter, we describe well-established and reproducible laboratory methods and protocols typically used to determine: (1) the ADP-ribose chain length(s) and (2) the molecular stoichiometry of the protein–poly(ADP-ribosyl)ation reaction, e.g., number of ADP-ribose chains/polypeptide unit. While the methodology described here is exclusively for in vitro purified systems that can be used with high reliability, the reader is advised that application of these protocols to whole cell extracts and tissue systems must take into consideration the rapid turnover rate of protein-bound ADP-ribose polymers in vivo. Indeed, these extremely low-abundance chromatin-bound polymeric molecules are notoriously characterized for displaying a short half-life, typically from a few seconds to a few minutes. We also discuss potential methodological pitfalls, such as: (1) the chemical stability of protein–(ADP-ribose)n adducts and (2) the requirement for polymeric radiolabeling.