All lanes : Anti-Cleaved PARP1 antibody [E51] (ab32064) at 1/10000 dilution

Lane 1 : Wild-type (1uM Staurosporine for 3hrs) HAP1 cell lysate
Lane 2 : Wild-type (Staurosporine control) HAP1 cell lysate
Lane 3 : PARP1 knockout (1uM Staurosporine for 3hrs) HAP1 cell lysate
Lane 4 : PARP1 knockout (Staurosporine control) HAP1 cell lysate
Lane 5 : HeLa (1uM Staurosporine for 3hrs) cell lysate
Lane 6 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 25 kDa
Observed band size: 27 kDa why is the actual band size different from the predicted?

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.

All lanes : Anti-Cleaved PARP1 antibody [E51] (ab32064) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysates treated with 1uM Staurosporine for 3 hours
Lane 2 : Untreated HeLa whole cell lysates
Lane 3 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysates treated with 1uM Staurosporine for 3 hours
Lane 4 : Untreated NIH/3T3 whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 25 kDa
Observed band size: 25 kDa

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).

All lanes : Anti-Cleaved PARP1 antibody [E51] (ab32064) at 1/10000 dilution

Lane 1 : Untreated Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate
Lane 2 : Jurkat cell lysate treated with camptothecin

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 25 kDa
Observed band size: 25 kDa

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

All lanes : Anti-Cleaved PARP1 antibody [E51] (ab32064) at 1/1000 dilution

Lane 1 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate
Lane 2 : NIH/3T3 (Mouse embryo fibroblast cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 25 kDa
Observed band size: 25 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Cleaved PARP1 antibody [E51] (ab32064) at 1/1000 dilution

Lane 1 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysates treated with 1uM Staurosporine for 3 hours
Lane 2 : Untreated PC-12 whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 25 kDa
Observed band size: 25 kDa


Exposure time: 30 seconds

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.