Anti PARP1 (N-term ZF1)  

Immunohistochemistry with anti-PARP-1 antibody showing nuclear positivity in human lung tissue at 20x and 40x (B & C). Staining was performed on Leica Bond system using the standard protocol. Formalin fixed/paraffin embedded tissue sections were subjected to antigen retrieval and then incubated with rabbit anti-PARP-1 antibody 200-401-GM8 at 1:100 dilution for 60 minutes. Biotinylated Anti-rabbit secondary antibody was used at 1:200 dilution to detect primary antibody. The reaction was developed using streptavidin-HRP conjugated compact polymer system and visualized with chromogen substrate, 3’3-diamino-benzidine substrate (DAB). The sections were then counterstained with hematoxylin to detect cell nuclei.

Western Blot of Rabbit anti-PARP1 antibody. Marker: Opal Pre-stained ladder (p/n MB-210-0500). Lane 1: HEK293 lysate (p/n W09-000-365). Lane 2: HeLa Lysate (p/n W09-000-364). Lane 3: MCF-7 Lysate (p/n W09-000-360). Lane 4: Jurkat Lysate (p/n W09-000-370). Lane 5: A431 Lysate (p/n W09-000-361). Lane 6: A549 Lysate (p/n W09-001-372). Lane 7: LNCap Lysate (p/n W09-001-GJ9). Lane 8: MOLT-4 Lysate (p/n W09-001-GK2). Lane 9: Ramos Lysate (p/n W09-000-GK4). Lane 10: Raji Lysate (p/n W09-001-368). Lane 11: A-172 Lysate (p/n W09-001-GL5). Lane 12: NIH/3T3 Lysate (p/n W10-000-358). Load: 35 μg per lane. Primary antibody: PARP1 antibody at 1ug/mL overnight at 4C. Secondary antibody: Peroxidase rabbit secondary antibody (p/n 611-103-122) at 1:30,000 for 60 min at RT. Blocking Buffer: 1% Casein-TTBS (p/n MB-082) for 30 min at RT. Predicted/Observed size: 113 kDa for PARP1.

Western Blot of recombinant PARP1 with rabbit anti-PARP1 (N-term ZF1) antibody. Lane 1: PARP1-Zinc Finger domain recombinant protein. Load: 0.05 μg per lane. Primary antibody: PARP1 (N-term ZF1) antibody at 1μg/mL for overnight at 4°C. Secondary antibody: HRP Gt-a-rabbit secondary antibody (p/n 611-103-122) at 1:40,000 for 30 min at RT. Block: MB-070 overnight at 4°C. Predicted/Observed size: 13 kDa for rPARP1 (N-term ZF1). Other band(s): none.

Western Blot of Rabbit anti-PARP1 N-term Antibody. Lane 1: Opal Pre-stained ladder (p/n MB-210-0500). Lane 2: OVCAR-8 Wild Type. Lane 3: PARP1-KO. Lane 4: PARP2-KO. Lane 5: PARP3-KO. Lane 6: PARP4-KO Lane 7: PARP5a-KO. Lane 8: PARP5b-KO. Lane 9: PARP6-KO. Lane 10: PARP7-KO. Lane 11: PARP8-KO. Lane 12: PARP9-KO. Lane 13: PARP10-KO. Lane 14: PARP12-KO. Lane 15: PARP13-KO. Lane 16: PARP14-KO. Lane 17: PARP16-KO. Load: 5.0 μg per lane. Primary antibody: PARP1 n-term antibody at 1ug/mL overnight at 4°C. Secondary antibody: Goat anti-rabbit Peroxidase secondary antibody (p/n 611-103-122) at 1:40,000 for 30 min at RT. Blocking Buffer: MB-073 for 30 min at RT. Predicted/Observed size: ~113 kDa for PARP1.

OVCAR-8 Wild Type Lysate was separated on 2D SDS-PAGE and blotted on PVDF to analyze immunocoverage of PARP1 antibody specific for the zinc finger 1 domain of PARP1. Primary Antibody: Anti-PARP1 (n-term) antibody 1:200 overnight at 4°C. Secondary Antibody: Goat anti-rabbit Peroxidase (611-103-122) at 1:2,000 at RT for 30min. Blocking Buffer: BlockOut (p/n MB-073) for 30 min at RT. Predicted/observed: ~110 kDa and pI 9.7.

Western Blot of endogenous PARP1 with Rabbit Anti-PARP1 (N-term ZF1) Antibody. Lane 1: OVCAR8 Wild Type lysate. Lane 2: OVCAR8 PARP1 KO lysate. Load: 5 μg per lane. Primary antibody: PARP1 (N-term ZF1) antibody at 1μg/mL for overnight at 4°C. Secondary antibody: HRP Gt-a-Rb IgG secondary antibody ( p/n 611-103-122) at 1:40,000 for 30 min at RT. Block: MB-070 overnight at 4°C. Predicted/Observed size: 113 kDa for endogenous PARP1. Other band(s): none. Image in collaboration with Phil Lorenzi at MD Anderson.

Western Blot of endogenous PARP1 with Rabbit Anti-PARP1 Antibodies. Lane 1: OVCAR8 Wild Type lysate. Lane 2: OVCAR8 PARP1 KO lysate. Load 5 μg per lane. Primary Antibody: Blot A: Anti-PARP1- n term (p/n 200-401-GM8); Blot B: Anti-PARP1- internal (p/n 200-401-X51) at 1μg/mL for overnight at 4°C. Secondary antibody: HRP Gt-a-Rb IgG secondary antibody (p/n 611-103-122) at 1:40,000 for 30 min at RT. Block: MB-070 overnight at 4°C. Predicted/Observed size: 113 kDa for endogenous PARP1. Other band(s): nonspecific ~ 40kDa in PARP1-AD only.

Peggy Sue？ Size Separation Electropherogram of OVCAR-8 lysates in no-salt buffer and detected with Anti-PARP1 (N-term ZF1). UV immobilization time: 250 seconds. Protein concentration: 577 μg/mL; 120 s UV immobilization. Primary antibody concentration: 20μg/mL. Primary antibody incubation time: 180 min. Exposure time: multi-image analysis exposure. Predicted/observed: ~116 kDa. Image courtesy of Phil Lorenzi at MD Anderson.