All lanes : Anti-RUNX1 / AML1 antibody [EPR23044-100] (ab240639) at 1/1000 dilution

Lane 1 : THP-1 (human monocytic leukemia monocyte), whole cell lysate
Lane 2 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate
Lane 3 : MOLT-4 (human lymphoblastic leukemia T lymphoblast), whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 48 kDa
Observed band size: 27-55 kDa why is the actual band size different from the predicted?

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

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

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.

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.

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.

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.