Anti-ARID1A antibody [EPR13501] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

Clone EPR13501 (ab217154) has been successfully conjugated by Abcam. This image was generated using Anti-ARID1A antibody [EPR13501] (Alexa Fluor® 488). Please refer to ab216112 for protocol details.

ab216112 staining ARID1A in wild-type HAP1 cells (top panel) and ARID1A knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab216112 at 1/500 dilution (shown in green) and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182560).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182560).

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

This data was developed using ab182560, the same antibody clone in a different buffer formulation.

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

This IHC data was generated using the same anti-ARID1A antibody clone, EPR13501, in a different buffer formulation (cat# 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.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182560).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182560).

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

Clone EPR13501 (ab217154) has been successfully conjugated by Abcam. This image was generated using Anti-ARID1A antibody [EPR13501] (Alexa Fluor® 647). Please refer to ab216304 for protocol details.

ab216304 staining ARID1A in wild-type HAP1 cells (top panel) and ARID1A knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab216304 at 1/500 dilution (shown in red) and ab195887 at 1/250 dilution (shown in green) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IP

Collaborator

Immunoprecipitation - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

This data was developed using ab182560, the same antibody clone in a different buffer formulation.

Immunoprecipitation of ARID1A in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 2μg 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.

false

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

This data was developed using ab182560, the same antibody clone in a different buffer formulation.

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] - BSA and Azide free (AB217154)

This data was developed using ab182560, the same antibody clone in a different buffer formulation.

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 (ab209101) 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] - BSA and Azide free (AB217154)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/arid1a-antibody-epr13501-ab182560'>ab182560</a>) 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='/en-us/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='/en-us/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='/en-us/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

false

• WB

Lab

Western blot - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/arid1a-antibody-epr13501-ab182560'>ab182560</a>) 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='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 130-270 kDa

false

Exposure time: 20s

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-ARID1A antibody [EPR13501] - BSA and Azide free (AB217154)

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. This data was developed using the same antibody clone in a different buffer formulation (ab182560).