Anti-PARP1 antibody [E78]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PARP1 antibody [E78] (AB32071)

ab32071 was shown to react with PARP1 in wild-type HEK293T cells in immunocytochemistry with loss of signal observed in PARP1 knockout cell line ab266598. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab32071 at 1/50 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor^® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss (LSM-880).

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-PARP1 antibody [E78] (AB32071)

HEK293T wild-type and PARP1 knockout cells (ab266598) were labelled with a green or violet, fluorescent dye, respectively. Wild-type and knockout cells were mixed in a 1 : 1 ratio, fixed in 4% PFA and permeabilized in 0.1% Triton X-100.

Antibody staining was quantified using the Attune NxT Flow Cytometer with representative images showing the staining intensity in the knockout population (pink histogram, dashed line) compared to the wild-type cells (green histogram, solid line). Histograms with dotted lines represent secondary antibody-only controls in both wild-type and knockout cells.

Antibody dilution : 0.1 μg/μL.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• WB

Supplier Data

Western blot - Anti-PARP1 antibody [E78] (AB32071)

ab32071 was shown to react with PARP1 in wild-type HEK293T cells in Western blot with loss of signal observed in PARP1 knockout cell line ab266598. Wild-type HEK293T and PARP1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab32071 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-PARP1 antibody [E78] (ab32071) at 1/1000 dilution

Lane 1:

Wild-type HEK293T lysate at 30 µg

Lane 2:

PARP1 knock-out HEK293T lysate at 30 µg

Lane 2:

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

Predicted band size: 113 kDa

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• WB

Unknown

Western blot - Anti-PARP1 antibody [E78] (AB32071)

pro-form : 116kDa; p85 caspases cleaved form : 85kDa; proteolysis cleaved fragments : 71kDa, 55kDa and 42kDa

All lanes:

Western blot - Anti-PARP1 antibody [E78] (ab32071) 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='/ko/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 [E78] (AB32071)

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 ab32071 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32071 was shown to specifically react with PARP1 when PARP1 knockout samples were used. Wild-type and PARP1 knockout samples were subjected to SDS-PAGE. ab32071 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PARP1 antibody [E78] (ab32071)

Predicted band size: 113 kDa

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• WB

CiteAb

Western blot - Anti-PARP1 antibody [E78] (AB32071)

PARP1 western blot using anti-PARP1 antibody [E78] ab32071. Publication image and figure legend from Lassalle, S., Zangari, J., et al., 2016, Oncotarget, PubMed 27036030.

ab32071 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab32071 please see the product overview.

Effect of miR-375 on proliferation and cancer drug responseA. Nthy-ori 3-1 cells were seeded and transfected with pre-miR-375 or pre-miR-CTL at 20pM for 24h and vandetanib was then added for 48h. Dead cells were stained with propidium iodide (red) before microscopic analysis. Pictures representative of four biological replicates. B. Quantification of propidium iodide positive Nthy-ori 3-1 cells. C. Nthy-ori 3-1 cells were seeded and transfected with pre-miR-375 or pre-miR-CTL at 20pM for 24h and vandetanib was then added for 48h. Quantification of ERK, AKT pathways and PARP cleavage was performed by immunoblot. Tubulin (TUBA) and actin B (ACTB) protein levels were used as loading controls. D. Nthy-ori 3-1 cells were seeded in 96-well plates and transfected with pre-miR-375 or pre-miR-CTL either at 6.25, 12.5, 25 pM for 24h and then treated with either 1.25, 2.5, 5μM vandetanib for 48h. Cell proliferation was evaluated using BrdU incorporation for 3h. Single doses and combination doses were analysed using Compusyn software and a Combination index/effect dot plot was generated. CI<1 values are indicative of synergism. E. TT cells were seeded and transfected with antagomiR-375 (anti-miR-375) or antagomiR-CTL (anti-miR-CTL) for 24h and vandetanib was then added for 48h. Quantification of propidium iodide positive TT cells.

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