Anti-PARP1 antibody [E102]

8 product images

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  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

  Lane 2: Jurkat (Human T cell leukemia T lymphocyte) treated with 1μM staurosporine for 4 hours whole cell lysate

  Secondary

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

  Predicted band size: 113 kDa

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  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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, Goat Anti-Rabbit IgG H&L (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.

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  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 (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 Human PARP1 knockout HEK-293T cell line ab266598 (knockout cell lysate Human PARP1 knockout HEK-293T 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 (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (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 (Human PARP1 knockout HEK-293T cell line ab266598)

  Performed under reducing conditions.

  Predicted band size: 113 kDa

  Observed band size: 113 kDa

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  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, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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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  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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.

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  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, Goat Anti-Rabbit IgG H&L (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).

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  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.

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  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] (Anti-alpha Tubulin antibody [DM1A] - Loading Control 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 Human PARP1 knockout A549 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

  Performed under reducing conditions.