Anti-ARID1A 抗体 [EPR13501]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ARID1A antibody [EPR13501] (AB182560)

Immunofluorescent analysis of SH-SY5Y cells labeling ARID1A with ab182560 at 1/500 and Goat anti rabbit IgG(Alexa Fluor®555) at 1/200. Image at the right stained with DAPI.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501] (AB182560)

Immunohistochemical analysis of formalin fixed paraffin embedded human kidney labelling ARID1A with ab182560 at a concentration of 1µg/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. ab182560 Anti-ARID1A antibody [EPR13501] 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501] (AB182560)

Immunohistochemical analysis of formalin fixed paraffin embedded human placenta labelling ARID1A with ab182560 at a concentration of 1µg/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. ab182560 Anti-ARID1A antibody [EPR13501] 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).

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-ARID1A antibody [EPR13501] (AB182560)

ab182560 was shown to react with ARID1A in wild-type HAP1 cells in immunocytochemistry with loss of signal observed in a ARID1A knockout cell line. 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 ab182560 at 1/2000 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ARID1A antibody [EPR13501] (AB182560)

ab182560 staining ARID1A in wild-type HAP1 cells (top panel) and ARID1A knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab182560 at 1/500 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ARID1A antibody [EPR13501] (AB182560)

Intracellular Flow Cytometry analysis of SH-SY5Y (human neuroblastoma) cells labeling ARID1A with purified ab182560 at 1/230 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501] (AB182560)

Immunohistochemical analysis of paraffin embedded Human adenocarcinoma of endometrium without ARID1A mutation (Left image) labeling ARID1A using ab182560 at 1/1000 dilution. Right image : Right picture : paraffine embedded human adenocarcinoma of endometrium with ARID1A mutation. A Ready to use HRP Polymer for Rabbit IgG (prediluted) was used as secondary. Counterstain : Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA, pH 9 was performed before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501] (AB182560)

Immunohistochemical analysis of paraffin embedded Human kidney tissue (Left image) labeling ARID1A using ab182560 at 1/1000 dilution. Right image : Right picture : paraffine embedded human clear cell carcinoma of kidney with ARID1A mutation. A Ready to use HRP Polymer for Rabbit IgG (prediluted) was used as secondary. Counterstain : Hematoxylin.

Heat mediated antigen retrieval with Tris-EDTA, pH 9 was performed before commencing with IHC staining protocol.

• IP

Collaborator

Immunoprecipitation - Anti-ARID1A antibody [EPR13501] (AB182560)

Immunoprecipitation of ARID1A in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 2 μg of ab182560 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

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.

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501] (AB182560)

Immunohistochemical analysis of formalin fixed paraffin embedded rat kidney labelling ARID1A with ab182560 at a concentration of 1/2500 dilution. The section was incubated with ab182560 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. A ready to use Rabbit specific IHC polymer detection kit HRP/DAB was used as a secondary. Sections were counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Citrate buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501] (AB182560)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse kidney labelling ARID1A with ab182560 at a concentration of 1/2500 dilution. The section was incubated with ab182560 for 10 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. A ready to use Rabbit specific IHC polymer detection kit HRP/DAB was used as a secondary. Sections were counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Citrate buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins.

• WB

Lab

Western blot - Anti-ARID1A antibody [EPR13501] (AB182560)

Blocking buffer and concentration : 5% NFDM/TBS

ARID1A has many mutations which typically generate truncated proteins that are highly prone to degradation. (PMID : 21614196, PMID : 29486633, PMID : 34429326).

All lanes:

Western blot - Anti-ARID1A antibody [EPR13501] (ab182560) at 1/1000 dilution

All lanes:

293T (Human embryonic kidney epithelial cell) whole cell lysate 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

Observed band size: 130-270 kDa

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Exposure time: 20s

• WB

Lab

Western blot - Anti-ARID1A antibody [EPR13501] (AB182560)

Lanes 1 - 3 : Merged signal (red and green). Green - ab182560 observed at 270 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab182560 was shown to react with ARID1A in wild-type HEK-293T cells in Western blot with loss of signal observed in ARID1A knockout cell line ab266189 (ARID1A knockout cell lysate ab257250). Wild-type HEK-293T and ARID1A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab182560 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 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-ARID1A antibody [EPR13501] (ab182560) at 1/1000 dilution

Lane 1:

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

Lane 2:

Western blot - Human ARID1A knockout HEK-293T cell lysate (<a href='/products/cell-lysates/human-arid1a-knockout-hek-293t-cell-lysate-ab257250'>ab257250</a>) at 20 µg

Lane 3:

SH-SY5Y cell lysate at 20 µg

Secondary

Lanes 1 - 3:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 3:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 242 kDa

Observed band size: 270 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-ARID1A antibody [EPR13501] (AB182560)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HCT116 cells and 5µg of ab182560 [EPR13501]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

CiteAb

Western blot - Anti-ARID1A antibody [EPR13501] (AB182560)

ARID1A western blot using anti-ARID1A antibody [EPR13501] ab182560. Publication image and figure legend from Zhao, B., Lin, J., et al., 2019, Nat Commun, PubMed 31492885.

ab182560 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 ab182560 please see the product overview.

ARID1A inactivation causes DNA damage at telomeres. a, b Schematic of synchronization and release (a) and immunoblot of DNA damage marker γH2AX (b) in parental and ARID1A knockout RMG1 cells. Phosphorylated Histone H3 at serine 10 (pH3S10) was used as a marker of mitosis. Relative intensities of immunoblot bands were quantified underneath. c, d Co-staining of telomere by FISH and γH2AX (c) and quantification of telomeric DNA damage (d) in mitotic parental and ARID1A knockout RMG1 cells after cytospin. e, f Co-staining of telomere by FISH and γH2AX in ARID1A-mutated TOV21G mitotic cells (e) and quantification of mitotic telomeric DNA damage in a panel of clear cell ovarian cancer cell lines or primary cultures highlighted in red (f). g, h Representative images (g) and quantification (h) of telomere DNA damage in parental, P53−/−/Pten−/− and P53−/−/Pten−/−/Arid1a−/− mouse bladder organoid cultures. n = 3 independent experiments unless otherwise stated. Data represent mean ± s.e.m. Scale bar = 10 μm. P values were calculated using a two-tailed t test except in 2f and 2h by multilevel mixed-effects models

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