Anti-RUNX1 / AML1 antibody [EPR23309-113] - ChIP Grade

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-RUNX1 / AML1 antibody [EPR23309-113] - ChIP Grade (AB272456)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized Jurkat (Human T cell leukemia T lymphocyte) cells labelling RUNX1 / AML1 with ab272456 at 1/500 dilution (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

Lab

Immunoprecipitation - Anti-RUNX1 / AML1 antibody [EPR23309-113] - ChIP Grade (AB272456)

RUNX1 / AML1 was immunoprecipitated from K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab272456 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab272456 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : K562 whole cell lysate 10 μg

Lane 2 : ab272456 IP in K562 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab272456 in K562 whole cell lysate

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : 3 mins.

All lanes:

Immunoprecipitation - Anti-RUNX1 / AML1 antibody [EPR23309-113] - ChIP Grade (ab272456)

Predicted band size: 48 kDa

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• IP

Unknown

Immunoprecipitation - Anti-RUNX1 / AML1 antibody [EPR23309-113] - ChIP Grade (AB272456)

RUNX1 / AML1 was immunoprecipitated from 0.35 mg Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate with ab272456 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab272456 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate 20 ug

Lane 2 : ab272456 IP in Jurkat whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab272456 in Jurkat whole cell lysate

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

Exposure time : 32 seconds.

All lanes:

Immunoprecipitation - Anti-RUNX1 / AML1 antibody [EPR23309-113] - ChIP Grade (ab272456)

Predicted band size: 48 kDa

Observed band size: 27-38 kDa

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• ChIP

Supplier Data

ChIP - Anti-RUNX1 / AML1 antibody [EPR23309-113] - ChIP Grade (AB272456)

Chromatin was prepared from K-562 cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 μg of chromatin, 5 μg of ab272456 (red), or 5 μg of rabbit normal IgG ab172730 (gray) and 20 μl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are from paper PMID：20959602

The RUNX1 enrichment profile is consistent with what have been described in literature (PMID : 20959602).

• WB

Lab

Western blot - Anti-RUNX1 / AML1 antibody [EPR23309-113] - ChIP Grade (AB272456)

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

RUNX1 has several isoforms. The molecular weight observed is consistent with what have been described in literature (PMID : 17431130, 29296779).

Exposure time : 3 minutes

All lanes:

Western blot - Anti-RUNX1 / AML1 antibody [EPR23309-113] - ChIP Grade (ab272456) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 2:

MOLT-4 (human lymphoblastic leukemia t lymphoblast) whole cell lysate at 20 µg

Lane 3:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 48 kDa

Observed band size: 27-48 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-RUNX1 / AML1 antibody [EPR23309-113] - ChIP Grade (AB272456)

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 ab272456 [EPR23309-113]. 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 Support and downloads section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.