Synthesis and Purification of PAR

According to Kiehlbauch et al. (19 (link)), PAR was synthesized with some modifications. Briefly, PAR was synthesized in a 20 ml incubation mixture comprising 100 mM Tris–HCl pH 7.8, 10 mM MgCl₂, 1 mM NAD⁺, 10 mM DTT, 60 μg/ml histone H1, 60 μg/ml histone type IIa, 50 μg/ml octameric ‘activator’ oligonucleotide GGAATTCC and 150 nM human PARP-1. The reaction was stopped after 15 min by addition of 20 ml ice-cold 20% TCA. Following precipitation the pellet was washed with ice-cold 99.8% ethanol. Polymer was detached using 0.5 M KOH/50 mM EDTA and purified as described (36 (link)). After extraction with phenol–chloroform–isoamyl alcohol PAR was precipitated with ethanol overnight. Following centrifugation PAR was air-dried and stored at −20°C