Recombinant Anti-RUNX3 antibody [EPR20687] - ChIP Grade (ab224641)

Lanes 1 - 4: Merged signal (red and green). Green - ab224641 observed at 44-46 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab224641 was shown to specifically react with RUNX3 in wild-type HAP1 cells as signal was lost in RUNX3 knockout cells. Wild-type and RUNX3 knockout samples were subjected to SDS-PAGE. Ab224641 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at  1/1000 dilution and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

Blocking/Dilution: 5% NFDM/TBST.

Negative control: MCF7 (PMID:21706051).

This target detects both predicted isoforms 44KDa and 46KDa that consistent with what has been described in the literature (PMID:23700080).

Immunohistochemical analysis of paraffin-embedded human stomach tissue labeling RUNX3 with ab224641 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use. Nuclear staining on lymphoid cells of human stomach (PMID:15514019; PMID:21786422) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use.

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Ramos (human Burkitt's lymphoma cell line) cells labeling RUNX3 with ab224641 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on Ramos cell line. DAPI (blue) and anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution were used as counterstains.

The negative controls are as follows:

Negative control: MCF7 (human breast adenocarcinoma cell line) (PMID: 21706051).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is AlexaFluor^®488 Goat anti-Rabbit (ab150077) at 1/1000 dilution..

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized Raji (human Burkitt's lymphoma cell line) cell line labeling RUNX3 with ab224641 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody. 

Chromatin was prepared from U-937 (PMA treated or not) cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab224641 (red), and 20µl of protein A/G sepharose beads slurry (10µl of sepharose A beads + 10µl of sepharose G beads). 5μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

RUNX3 was immunoprecipitated from 0.35 mg of Raji (human Burkitt's lymphoma cell line) whole cell lysate with ab224641 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab224641 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Lane 1: Raji whole cell lysate 10 µg (Input). 

Lane 2: ab224641 IP in Raji whole cell lysate (+). 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab224641 in Raji whole cell lysate (-).

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

Blocking/Dilution: 5% NFDM/TBST.

This target detects both predicted isoforms 44KDa and 46KDa that consistent with what has been described in the literature (PMID:23700080).

Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling RUNX3 with ab224641 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use. Nuclear staining on lymphoid cells of human gastric cancer (PMID:27566570) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use.

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji (human Burkitt's lymphoma cell line) cells labeling RUNX3 with ab224641 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on Raji cell line. DAPI (blue) and anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution were used as counterstains.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is AlexaFluor^®488 Goat anti-Rabbit (ab150077) at 1/1000 dilution.

Immunohistochemical analysis of paraffin-embedded human diffuse large B cell lymphoma tissue labeling RUNX3 with ab224641 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use. Nuclear staining on human diffuse large B cell lymphoma (PMID:27184221) is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051), ready to use.

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).