Synthesis of PAR was performed as previously reported20 (link). Briefly, 50 units of purified human PARP-1 (High Specific Activity hPARP-1, Trevigen) were incubated in a mixture containing 100 mM Tris-HCl pH 8, 10 mM MgCl2, 2 mM dithiothreitol, 2.5 μg of DNase I-activated calf thymus DNA (Trevigen) and 200 mM NAD+ (Sigma-Aldrich) for 45 min at 30 °C. The reaction was stopped by adding ice-cold trichloroacetic acid (TCA) to a final concentration of 20% (w/v). PARs were detached from proteins by incubation in 50 mM NaOH and 10 mM EDTA for 1 hr at 60 °C. After adjustment of pH to 7.5, PAR were purified by phenol/chloroform extraction as described20 (link).
For the study of non-covalent interaction of PAR with TRF-1, graded concentrations of purified His-hTRF1 protein were immobilized directly by slot-blotting on nitrocellulose membranes. Histone H1 (Millipore) was used as positive control in the PAR binding assay. Subsequently, filters were incubated with PAR diluted in TBS-T (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 1 hr at room temperature. After high-stringency salt washes, protein bound PAR were detected using the anti-PAR monoclonal antibody (mouse Mab ALX-804220).
For the study of non-covalent interaction of PAR with TRF-1, graded concentrations of purified His-hTRF1 protein were immobilized directly by slot-blotting on nitrocellulose membranes. Histone H1 (Millipore) was used as positive control in the PAR binding assay. Subsequently, filters were incubated with PAR diluted in TBS-T (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 1 hr at room temperature. After high-stringency salt washes, protein bound PAR were detected using the anti-PAR monoclonal antibody (mouse Mab ALX-804220).
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