Anti-ARID1A antibody [EPR13501-73] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ARID1A antibody [EPR13501-73] - BSA and Azide free (AB250617)

This data was developed using ab182561, the same antibody clone in a different buffer formulation.

Intracellular Flow Cytometry analysis of SH-SY5Y (human neuroblastoma) cells labeling ARID1A with purified ab92512 at 1/500 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 [EPR13501-73] - BSA and Azide free (AB250617)

This data was developed using ab182561, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue (left image) labeling ARID1A using ab182561 at 1/1000 dilution. Right image : 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-ARID1A antibody [EPR13501-73] - BSA and Azide free (AB250617)

This data was developed using ab182561, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of SH-SY5Y cells labeling ARID1A with ab182561 at 1/500 and Goat anti rabbit IgG(Alexa Fluor®555) at 1/200. Image at the right stained with DAPI.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501-73] - BSA and Azide free (AB250617)

This data was developed using ab182561, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human adenocarcinoma of endometrium without ARID1A mutation (left image) labeling ARID1A using ab182561 at 1/1000 dilution. Right image : human adenocarcinoma of endometrium 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.

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-ARID1A antibody [EPR13501-73] - BSA and Azide free (AB250617)

This data was developed using ab182561, the same antibody clone in a different buffer formulation.

ab182561 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 ab182561 at 1/250 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.

• IP

Collaborator

Immunoprecipitation - Anti-ARID1A antibody [EPR13501-73] - BSA and Azide free (AB250617)

This data was developed using ab182561, the same antibody clone in a different buffer formulation.

Immunoprecipitation of ARID1A in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 2 μg of ab182561 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 [EPR13501-73] - BSA and Azide free (AB250617)

This data was developed using ab182561, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat spleen tissue labeling ARID1A with ab182561 at 1/4000 dilution followed by a Goat Anti-Rabbit IgG H&L (HRP polymer) - ready to use.

Positive staining on mouse spleen.

The primary antibody was incubated at 4°C overnight.

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ARID1A antibody [EPR13501-73] - BSA and Azide free (AB250617)

This data was developed using ab182561, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling ARID1A with ab182561 at 1/4000 dilution followed by a Goat Anti-Rabbit IgG H&L (HRP polymer) - ready to use.

Positive staining on mouse spleen.

The primary antibody was incubated at 4°C overnight.

Counterstained with Hematoxylin.

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0).

• WB

Supplier Data

Western blot - Anti-ARID1A antibody [EPR13501-73] - BSA and Azide free (AB250617)

This data was developed using ab182561, the same antibody clone in a different buffer formulation. Blocking and diluting buffer : 5% NFDM/TBST 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 [EPR13501-73] (<a href='/en-us/products/primary-antibodies/arid1a-antibody-epr13501-73-ab182561'>ab182561</a>) at 1/1000 dilution

Lane 1:

ARID1 knockout HAP1 (Human chronic myelogenous leukemia near-haploid cell line) treated with 10uM MG-132 for 6 hours whole cell lysate at 20 µg

Lane 2:

Wild-type HAP1 (Human chronic myelogenous leukemia-haploid cell line) treated with 10uM MG-132 for 6 hours 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

Predicted band size: 242 kDa

Observed band size: 130-270 kDa

false

Exposure time: 10s

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-ARID1A antibody [EPR13501-73] - BSA and Azide free (AB250617)

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 ab182561 [EPR13501-73]. 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 ab182561, the same antibody clone in a different buffer formulation.

• WB

Lab

Western blot - Anti-ARID1A antibody [EPR13501-73] - BSA and Azide free (AB250617)

This data was developed using ab182561, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000,000 dilution.

This antibody failed to work in kidney tissues from mice and rats. 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 [EPR13501-73] (<a href='/en-us/products/primary-antibodies/arid1a-antibody-epr13501-73-ab182561'>ab182561</a>) at 1/1000 dilution

Lane 1:

Mouse spleen tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Rat spleen tissue lysate at 20 µg

Lane 5:

Rat kidney tissue lysate at 20 µg

Lane 6:

Rat brain tissue lysate at 20 µg

Lane 7:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 8:

C6 (rat glial tumor glial 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: 50-270 kDa,36 kDa

false

Exposure time: 180s