Anti-RUNX1 / AML1 antibody [EPR23044-100]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RUNX1 / AML1 antibody [EPR23044-100] (AB240639)

Immunohistochemical analysis of paraffin-embedded Human breast tissue labeling RUNX1 / AML1 with ab240639 at 1/2000 dilution (0.25 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human breast tissue is observed. The section was incubated with ab240639 for 15 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-RUNX1 / AML1 antibody [EPR23044-100] (AB240639)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Jurkat (Human T cell leukemia T lymphocyte) cells labelling RUNX1 / AML1 with ab240639 at 1/100 dilution, followed by ab240639 anti-RUNX1/AML1 ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing strong nuclear and weak cytoplasmic staining in Jurkat cell line is observed. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab240639 anti-RUNX1/AML1 ab150077 AlexaFluor®488 Goat anti-Rabbit secondary at 1/1000 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RUNX1 / AML1 antibody [EPR23044-100] (AB240639)

Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling RUNX1 / AML1 with ab240639 at 1/2000 dilution (0.25 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human breast carcinoma (PMID : 24967588) tissue is observed. The section was incubated with ab240639 for 15 mins at room temperature. The immunostaining staining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-RUNX1 / AML1 antibody [EPR23044-100] (AB240639)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Jurkat (Human T cell leukemia T lymphocyte) cells labelling RUNX1 / AML1 with ab240639 at 1/500 (Red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-RUNX1 / AML1 antibody [EPR23044-100] (AB240639)

RUNX1 / AML1 was immunoprecipitated from 0.35 mg Jurkat (human T cell leukemia T lymphocyte) whole cell lysate 10ug with ab240639 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab240639 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution. Lane 1 : Jurkat (human T cell leukemia T lymphocyte) whole cell lysate 10ug

Lane 2 : ab240639 IP in Jurkat whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab240639 in Jurkat whole cell lysate

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

Exposure time : 10 seconds

All lanes:

Immunoprecipitation - Anti-RUNX1 / AML1 antibody [EPR23044-100] (ab240639)

Predicted band size: 48 kDa

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• WB

Lab

Western blot - Anti-RUNX1 / AML1 antibody [EPR23044-100] (AB240639)

Western blot : Rabbit Monoclonal [EPR23044-100] to RUNX1 / AML1 ab240639 staining at a 1/200 dilution, shown in black; Mouse anti-CANX (ab238078) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 50-54 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in RUNX1 knockout U-87 MG cell line.

To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit HRP (H+L) & Goat anti-Mouse 680RD at 1/20,000 dilution.

Lanes 1 - 4:

Western blot - Anti-RUNX1 / AML1 antibody [EPR23044-100] (ab240639) at 1/200 dilution

Lanes 1 - 4:

Western blot - Anti-RUNX1 / AML1 antibody [EPR23044-100] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/runx1-aml1-antibody-epr23044-100-bsa-and-azide-free-ab264471'>ab264471</a>) at 1/200 dilution

Lane 1:

Wild-type U-87 MG at 40 µg

Lane 2:

Western blot - Human RUNx1 knockout U-87 MG cell line (ab306773) at 40 µg

Lane 3:

Jurkat at 20 µg

Lane 4:

Caco-2 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit HRP (H+L) & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 48 kDa

Observed band size: 50-54 kDa

true

Exposure time: 10s

• WB

Lab

Western blot - Anti-RUNX1 / AML1 antibody [EPR23044-100] (AB240639)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 23352661, 29296779). The RUNX1 gene has several isoforms, 3 major isotypes. RUNX1b is broadly expressed, and RUNX1a overexpression has been reported in AML.

Exposure time : Lane 1 : 48 seconds Lanes 2-3 : 26 seconds

All lanes:

Western blot - Anti-RUNX1 / AML1 antibody [EPR23044-100] (ab240639) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte), whole cell lysate at 10 µg

Lane 2:

Jurkat (human T cell leukemia T lymphocyte), whole cell lysate at 10 µg

Lane 3:

MOLT-4 (human lymphoblastic leukemia T lymphoblast), whole cell lysate at 10 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: 48 kDa

Observed band size: 27-55 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-RUNX1 / AML1 antibody [EPR23044-100] (AB240639)

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 K-562 (Human chronic myelogenous leukemia lymphoblast) cells and 5µg of ab240639 [EPR23044-100]. 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.