Anti-Cleaved PARP1 抗体 [E51]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cleaved PARP1 antibody [E51] (AB32064)

Immunohistochemical analysis of formalin fixed paraffin embedded human ovarian carcinoma labelling Cleaved PARP1 with ab32064 at a concentration of 0.5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins. ab32064 anti-Cleaved PARP1 antibody [E51] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cleaved PARP1 antibody [E51] (AB32064)

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cleaved PARP1 antibody [E51] (AB32064)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cleaved PARP1 antibody [E51] (AB32064)

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).

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-Cleaved PARP1 antibody [E51] (ab32064) at 1/10000 dilution

Lane 1:

Wild-type (1uM Staurosporine for 3hrs) HAP1 cell lysate at 20 µg

Lane 2:

Wild-type (Staurosporine control) HAP1 cell lysate at 20 µg

Lane 3:

PARP1 knockout (1uM Staurosporine for 3hrs) HAP1 cell lysate at 20 µg

Lane 4:

PARP1 knockout (Staurosporine control) HAP1 cell lysate at 20 µg

Lane 5:

HeLa (1uM Staurosporine for 3hrs) cell lysate at 20 µg

Lane 6:

HeLa cell lysate at 20 µg

Predicted band size: 113 kDa

Observed band size: 27 kDa

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• WB

Lab

Western blot - Anti-Cleaved PARP1 antibody [E51] (AB32064)

Western blot : Anti-PARP1 antibody [E51] (ab32064) staining at 1/10000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab32064 was shown to bind specifically to PARP1. A band was observed at 27 kDa in wild-type A549 cell lysates with no signal observed at this size in PARP1 knockout cell line. To generate this image, wild-type and PARP1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Cleaved PARP1 antibody [E51] (ab32064) at 1/10000 dilution

Lane 1:

Wild-type A549 control staurosporine (0 uM, 72 h) cell lysate at 20 µg

Lane 2:

Wild-type A549 treated staurosporine (3 uM, 24 h) cell lysate at 20 µg

Lane 3:

Wild-type A549 control staurosporine (3 uM, 72 h) cell lysate at 20 µg

Lane 4:

PARP1 knockout A549 treated staurosporine (3 uM, 24 h) cell lysate at 20 µg

Observed band size: 27 kDa

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• WB

Lab

Western blot - Anti-Cleaved PARP1 antibody [E51] (AB32064)

Blocking/Dilution buffer 5% NFDM/TBST

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

All lanes:

Western blot - 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 at 20 µg

Lane 2:

Untreated HeLa whole cell lysates at 20 µg

Lane 3:

NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysates treated with 1uM Staurosporine for 3 hours at 20 µg

Lane 4:

Untreated NIH/3T3 whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 113 kDa

Observed band size: 25 kDa

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• WB

Lab

Western blot - Anti-Cleaved PARP1 antibody [E51] (AB32064)

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.

All lanes:

Western blot - Anti-Cleaved PARP1 antibody [E51] (ab32064)

Predicted band size: 113 kDa

Observed band size: 25 kDa

true

Exposure time: 10s

• WB

Lab

Western blot - Anti-Cleaved PARP1 antibody [E51] (AB32064)

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:

Western blot - Anti-Cleaved PARP1 antibody [E51] (ab32064)

Predicted band size: 113 kDa

false

• WB

Lab

Western blot - Anti-Cleaved PARP1 antibody [E51] (AB32064)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - 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 at 10 µg

Lane 2:

Jurkat cell lysate treated with camptothecin at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 113 kDa

Observed band size: 25 kDa

false

• WB

Unknown

Western blot - Anti-Cleaved PARP1 antibody [E51] (AB32064)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - 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 at 20 µg

Lane 2:

NIH/3T3 (Mouse embryo fibroblast cell line) cell lysate at 20 µg

Secondary

All lanes:

HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 113 kDa

Observed band size: 25 kDa

false

• WB

Lab

Western blot - Anti-Cleaved PARP1 antibody [E51] (AB32064)

Blockinng/Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - 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 at 20 µg

Lane 2:

Untreated PC-12 whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 113 kDa

Observed band size: 25 kDa

false

Exposure time: 30s