Anti-PARP1 antibody [E102]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PARP1 antibody [E102] (AB32138)

Flow cytometry overlay histogram showing wild-type Hap1 (green line) and PARP1 knockout Hap1 stained with ab32138 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab32138) (1x 106 in 100μl at 0.04 μg/ml (1/55750)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hap1 - black line, PARP1 knockout Hap1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PARP1 antibody [E102] (AB32138)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling PARP1 with purified ab32138 at 1/200 dilution (0.51 µg/mL). Heat mediated antigen retrieval was performed using Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PARP1 antibody [E102] (AB32138)

Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PARP1 with purified ab32138 at 1/100 dilution (1.0 μg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% TritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PARP1 antibody [E102] (AB32138)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling PARP1 with purified ab32138 at 1/20 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• WB

Supplier Data

Western blot - Anti-PARP1 antibody [E102] (AB32138)

pro-form : 116kDa; p25 caspases cleaved form : 25kDa; proteolysis cleaved fragments : 58kDa and 42kDa

All lanes:

Western blot - Anti-PARP1 antibody [E102] (ab32138) at 1/1000 dilution

Lane 1:

Untreated Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

Jurkat (Human T cell leukemia T lymphocyte) treated with 1μM staurosporine for 4 hours whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 113 kDa

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• WB

Lab

Western blot - Anti-PARP1 antibody [E102] (AB32138)

Western blot : Anti-PARP1 antibody [E102] (ab32138) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab32138 was shown to bind specifically to PARP1. A band was observed at 125 kDa in wild-type A549 cell lysates with no signal observed at this size in PARP1 knockout cell line ab276094 (knockout cell lysate Abcam Pools). 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 5 % 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-PARP1 antibody [E102] (ab32138) at 1/1000 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:

PARP1 knockout A549 control staurosporine (0 uM, 72 h) cell lysate at 20 µg

Lane 4:

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

Lane 5:

Empty cell lysate at 20 µg

Lane 6:

HAP1 Treated Staurosporine (2uM, 4h) cell lysate at 20 µg

Lane 7:

HAP1 Vehicle Control Staurosporine (0uM, 4h) cell lysate at 10 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

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• WB

Lab

Western blot - Anti-PARP1 antibody [E102] (AB32138)

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 - ab32138 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32138 was shown to specifically react with PARP1 when PARP1 knockout samples were used. Wild-type and PARP1 knockout samples were subjected to SDS-PAGE. ab32138 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10 000 dilution respectively. Blots were developed with 800CW Goat anti Rabbit and 680CW Goat anti Mouse secondary antibodies at 1/10 000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PARP1 antibody [E102] (ab32138)

Predicted band size: 113 kDa

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• WB

Lab

Western blot - Anti-PARP1 antibody [E102] (AB32138)

Lanes 1- 2 : Merged signal (red and green). Green - ab32138 observed at 113 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32138 was shown to react with PARP1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266598 (knockout cell lysate ab257017) was used. Wild-type HEK-293T and PARP1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab32138 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PARP1 antibody [E102] (ab32138) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

PARP1 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human PARP1 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-parp1-knockout-hek-293t-cell-line-ab266598'>ab266598</a>)

Predicted band size: 113 kDa

Observed band size: 113 kDa

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