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Peggy SueTM Size Separation Electropherogram of OVCAR 8 lysates in no salt buffer and detected with Anti PARP1 (internal). UV immobilization time: 250 seconds. Protein concentration: 577 ug/mL; 120 s UV immobilization. Primary antibody concentration: 20ug/mL. Primary antibody incubation time: 180 min. Exposure time: 10 seconds. Predicted/observed: ~116 kDa. Image courtesy of Phil Lorenzi at MD Anderson.
OVCAR-8 Wild Type Lysate separated on 2D SDS-PAGE and blotted on PVDF to analyze immunocoverage of PARP1 antibody specific for the autocatalytic domain of PARP1. Primary Antibody: Anti-PARP1 (internal) antibody 1:200 overnight at 4C. Secondary Antibody: Goat anti-rabbit Peroxidase (p/n 611-103-122) at 1:2,000 at RT for 30min. Blocking Buffer: BlockOut (p/n MB-073) for 30min at RT. Predicted/observed: ~110 kDa and pI 9.7. Other spots detected: cleavage products of PARP1.
Western Blot of Rabbit Anti-PARP1 (internal) Antibody. Lane 1: OVCAR8 Wild Type lysate. Lane 2: OVCAR8 PARP1 KO lysate. Load: 5 ug per lane. Primary antibody: PARP1 (internal) antibody at 1:1000 for overnight at 4C. 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 4C. Predicted/Observed size: ~113kDa endogenous for PARP1. Other band(s): nonspecific ~ 40kDa.
Western Blot of recombinant PARP1 with Rabbit anti-PARP1 (internal) antibody. Lane 1: PARP1-autocatalytic domain recombinant protein. Load: 0.05 ug per lane. Primary antibody: PARP1 (internal) antibody at 1ug/mL for overnight at 4C. 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 4C. Predicted/Observed size: ~19 kDa for rPARP1 (internal) Other band(s): none.
Western Blot of Rabbit anti-PARP1 antibody. Lane 1: Molecular Weight ladder. 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 ug per lane. Primary antibody: PARP1 antibody at 1ug/mL overnight at 4C. 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 size: ~113kDa for PARP1. Observed nonspecific ~40kDa.
Western Blot of endogenous PARP1 with Rabbit Anti-PARP1 Antibodies.Lane 1: OVCAR8 Wild Type lysate. Lane 2: OVCAR8 PARP1 KO lysate.Load 5 ug 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 1ug/mL for overnight at 4C.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 4C.Predicted/Observed size: 113 kDa for endogenous PARP1. Other band(s): nonspecific ~ 40kDa in PARP1-AD only.
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