Anti-ARID1A antibody [EPR13501]

9 product images

• 

  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.

• 

  Western blot - Anti-ARID1A antibody [EPR13501] (ab182560)

  Lanes 1 - 3: Merged signal (red and green). Green - ab182560 observed at 270 kDa. Red - loading control Anti-GAPDH antibody [6C5] - 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 Human ARID1A knockout HEK-293T cell line ab266189 (ARID1A knockout cell lysate Human ARID1A knockout HEK-293T 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed 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: ARID1A knockout HEK-293T cell lysate at 20 µg

  Lane 2: Western blot - Human ARID1A knockout HEK-293T cell line (Human ARID1A knockout HEK-293T cell line ab266189)

  Lane 3: SH-SY5Y cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 242 kDa

  Observed band size: 270 kDa

• 

  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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

• 

  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.
  

• 

  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.

• 

  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.

• 

  Western blot - Anti-ARID1A antibody [EPR13501] (ab182560)

  Blocking buffer and concentration: 5% NFDM/TBSObserved band: 130-270 kDaARID1A 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 130 kDa, 270 kDa

  Exposure time: 20s

• 

  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 Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.
  
  Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.
  
  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• 

  Image collected and cropped by CiteAb under a CC-BY license from the publication

  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