Recombinant Anti-Cleaved PARP1 antibody [E51] (ab32064)

Lanes 1 - 6: Merged signal (red and green). Green - ab32064 observed at 27 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab32064 was shown to react with Cleaved PARP1 in wild-type HAP1 cells in Western blot with loss of signal observed in PARP1 knockout sample.Wild-type HAP1 and PARP1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab32064 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

Immunohistochemical staining of paraffin embedded rat colon with purified ab32064 at a working dilution of 1/100. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control (inset).

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: PARP1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: MCF7 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab32064 observed at 30 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32064 was shown to specifically react with PARP1 when PARP1 knockout samples were used. Wild-type and PARP1 knockout samples were subjected to SDS-PAGE. Ab32064 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilutions.

Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) ab216773 and 680CW Goat anti Mouse secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Blocking/Dilution buffer 5% NFDM/TBST

Exposure time :
Lane 1,2: 1 second
Lane 3,4: 8 seconds

Immunohistochemical staining of paraffin embedded human breast carcinoma tissue with unpurified ab32064 at a 1/100 dilution.

Immunohistochemical staining of paraffin embedded human ovarian carcinoma with purified ab32064 at a working dilution of 1 in 100. The secondary antibody used is a HRP polymer for rabbit IgG. Counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.

PBS was used instead of the primary antibody as the negative control (inset).

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Blocking/Dilution buffer: 5% NFDM/TBST.

Blockinng/Diluting buffer 5% NFDM/TBST

Jurkat (Human T cell leukemia cell line from peripheral blood) cells were incubated at 37°C for 24 hours with vehicle control (0 μM) and different concentrations of 15-Acetoxyscirpenol (ab142381). Increased expression of cleaved PARP1 (ab32064) in Jurkat cells correlates with an increase in 15-Acetoxyscirpenol concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab32064 at 1/10000 dilution and ab8227 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 and visualised using ECL development solution.