Anti-RUNX3 antibody [EPR20687] - ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (AB224641)

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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (AB224641)

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).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (AB224641)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (AB224641)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (AB224641)

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).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (AB224641)

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..

• IP

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Immunoprecipitation - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (AB224641)

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.

All lanes:

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

Predicted band size: 44 kDa

false

Exposure time: 1s

• ChIP

Unknown

ChIP - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (AB224641)

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).

• WB

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Western blot - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (AB224641)

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).

All lanes:

Western blot - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (ab224641) at 1/1000 dilution

Lane 1:

Raji (human Burkitt's lymphoma cell line) cell lysate at 20 µg

Lane 2:

Ramos (human Burkitt's lymphoma cell line) cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma cell line) 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 1/100000 dilution

Predicted band size: 44 kDa

Observed band size: 44 kDa,46 kDa

true

Exposure time: 5s

• WB

Supplier Data

Western blot - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (AB224641)

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).

All lanes:

Western blot - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (ab224641) at 1/1000 dilution

All lanes:

Human tonsil tissue 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 1/100000 dilution

Predicted band size: 44 kDa

true

Exposure time: 30s

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-RUNX3 antibody [EPR20687] - ChIP Grade (ab224641) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

RUNX3 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

Ramos whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate at 20 µg

Predicted band size: 44 kDa

false