Anti poly adp ribose monoclonal antibody clone 10h

PARylated PARP1 and purified PARylated PARP1-GCSF conjugate (2 μg of protein) were boiled with 10 mM DTT in NuPAGE LDS sample buffer (Thermo Fisher Scientific) at 98°C for 5 minutes. Samples were then run on 4–20% ExpressPlus-PAGE gels (GenScript, Piscataway, NJ), transferred to immun-blot PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were subsequently blocked with 5% BSA in PBS with 0.1% Tween-20 for 1 hour at room temperature, followed by incubation with appropriate primary antibodies (anti-poly-ADP-ribose (PAR) monoclonal antibody (clone: 10H) from Santa Cruz Biotechnology, and anti-GCSF (clone: BVD13–3A5) from BioLegend) and secondary antibodies (anti-mouse IgG-HRP (catalog: 62–6520), and anti-rat IgG-HRP (catalog # 31470) from Thermo Fisher Scientific). The immunoblots were developed by additions of supersignal west pico PLUS chemiluminescent substrate (Thermo Fisher Scientific) and imaged with a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc.).

PARP1, PARylated PARP1, and PARylated PARP1-Herceptin/UCHT1 (2 μg of protein) were boiled with 10 mM DTT in NuPAGE LDS sample buffer (Thermo Fisher Scientific) at 98°C for 5 minutes. Samples were then run on 4–20% ExpressPlus-PAGE gels (GenScript, Piscataway, NJ), transferred to immun-blot PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were subsequently blocked with 5% bovine serum albumin (BSA) in PBS with 0.1% Tween-20 (PBST) for 1 hour at room temperature, followed by incubation with anti-poly-ADP-ribose (PAR) monoclonal antibody (clone: 10H, Santa Cruz Biotechnology) and anti-mouse IgG-HRP (catalog: 62-6520, Thermo Fisher Scientific) or anti-human IgG (H+L)-HRP (catalog: 5220-0277, SeraCare). The immunoblots were developed by additions of supersignal west pico PLUS chemiluminescent substrate (Thermo Fisher Scientific) and imaged with a ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc).