Anti-pan Brd4 antibody [EPR25424-71]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized K-562 (human chronic myelogenous leukemia lymphoblast) cells labelling pan Brd4 with ab314432 at 1/500 (0.94 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1004 (2ug/ml) dilution (Green). Confocal image showing nuclear staining in K-562 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1004 (2ug/ml) dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized K-562 (human chronic myelogenous leukemia lymphoblast) cells labelling pan Brd4 with ab314432 at 1/500 dilution (0.1 ug, 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, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling pan Brd4 with ab314432 at 1/2000 (0.235 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human testis. The section was incubated with ab314432 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Immunohistochemical analysis of paraffin-embedded human ovarian cancer tissue labeling pan Brd4 with ab314432 at 1/2000 (0.235 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human ovarian cancer (PMID : 32044108). The section was incubated with ab314432 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• ChIP-seq

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ChIP-sequencing - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Chromatin was prepared from K562 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 8 µg of ab314432 [EPR25424-71]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• IP

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Immunoprecipitation - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

pan Brd4 was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab314432 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314432 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate Lane 2 : ab314432 IP in K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314432 in K-562 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-pan Brd4 antibody [EPR25424-71] (ab314432) at 1/30 dilution

All lanes:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 32s

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Chromatin was prepared from K562 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 8 µg of ab314432 [EPR25424-71]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Supplier Data

ChIP-sequencing - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Chromatin was prepared from K562 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 8 µg of ab314432 [EPR25424-71]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling pan Brd4 with ab314432 at 1/500 (0.94 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1005 (2ug/ml) dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cell line. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1005 (2ug/ml) dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling pan Brd4 with ab314432 at 1/2000 (0.235 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on rat testis. The section was incubated with ab314432 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling pan Brd4 with ab314432 at 1/2000 (0.235 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse testis. The section was incubated with ab314432 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling pan Brd4 with ab314432 at 1/500 dilution (0.1 ug, 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, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

pan Brd4 was immunoprecipitated from 0.35 mg ES-D3 [D3]  (mouse blastocyst-derived embryonic stem cell) whole cell lysate with ab314432 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314432 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : ES-D3 [D3]  (mouse blastocyst-derived embryonic stem cell) whole cell lysate Lane 2 : ab314432 IP in ES-D3 [D3]  (mouse blastocyst-derived embryonic stem cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab314432 in ES-D3 [D3] whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-pan Brd4 antibody [EPR25424-71] (ab314432) at 1/30 dilution

All lanes:

ES-D3 [D3]  (mouse blastocyst-derived embryonic stem cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 5s

• WB

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Western blot - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.0

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

The identity of the bands between 37 kDa and 100 kDa are unknown.

Exposure time : Lanes 1-3 : 81 seconds; Lane 4 : 3 seconds; Lane 5 : 81 seconds.

All lanes:

Western blot - Anti-pan Brd4 antibody [EPR25424-71] (ab314432) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 4:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 5:

ES-D3 D3  (mouse blastocyst-derived embryonic stem cell) whole 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

Observed band size: 110 kDa,200 kDa

false

• WB

Lab

Western blot - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Western blot : Rabbit Monoclonal [EPR25424-71] to Brd4 ab314432 staining at 1/1000 dilution, shown in green; Mouse anti alpha Tubulin (ab7291) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 200 kDa. 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-pan Brd4 antibody [EPR25424-71] (ab314432) at 1/1000 dilution

Lane 1:

Wild-type HAP1 (whole lysate) at 20 µg

Lane 2:

Wild-type HAP1 (Nuclear fraction) at 20 µg

Lane 3:

Wild-type HAP1 (Membrane fraction) at 20 µg

Lane 4:

HEK-293T (Whole lysate) at 20 µg

Lane 5:

HeLa (Whole lysate) at 20 µg

Lane 6:

HeLa (Nuclear fraction) at 20 µg

Lane 7:

HeLa (Membrane fraction) at 20 µg

Secondary

Lanes 1 - 7:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 7:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 200 kDa

Observed band size: 200 kDa,50 kDa

false

• WB

Supplier Data

Western blot - Anti-pan Brd4 antibody [EPR25424-71] (AB314432)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution. In Western blot, Anti-Brd4L antibody [EPR25425-23] (ab289886) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-pan Brd4 antibody [EPR25424-71] (ab314432) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

293T transfected with siRNA specifically targeti Brd4 whole 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

Observed band size: 110 kDa,200 kDa

false

Exposure time: 180s