Anti-BRD3 antibody [EPR30249-10]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling BRD3 with ab325539 at 1/100 (5.12 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized wildtype HEK-293T(human embryonic kidney epithelial cell); BRD3 knockout HEK-293T, ab266793 cells labelling BRD3 with ab325539 at 1/1000 (0.512 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing nuclear staining in wildtype HEK-293T cells (shown in green), showing no staining in BRD3 knockout HEK-293T cells. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type HEK293T (human embryonic kidney epithelial cell) cell pellet and (B) BRD3 knockout HEK293T cell pellet labeling BRD3 with ab325539 at 1/1000 (0.512 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) Wild-type HEK293T cell pellet, no staining on (B) BRD3 knockout HEK293T cell pellet. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Immunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue labeling BRD3 with ab325539 at 1/100 (5.12 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human colon carcinoma. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Immunohistochemical analysis of paraffin-embedded Human cervical carcinoma tissue labeling BRD3 with ab325539 at 1/100 (5.12 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human cervical carcinoma. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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

Lab

ChIP-sequencing - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Chromatin was prepared from K-562 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 8 µg of ab325539 [EPR30249-10]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Lab

ChIP-sequencing - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Chromatin was prepared from K-562 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 8 µg of ab325539 [EPR30249-10]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Lab

ChIP-sequencing - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Chromatin was prepared from K-562 cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 10^7 cells and 8 µg of ab325539 [EPR30249-10]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling BRD3 with ab325539 at 1/1000 (0.512 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing nuclear staining in Neuro-2a cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 (1 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

-ve control 1 : ab325539 at 1/1000 dilution, followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution, followed by ab150081 at 1/1000 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Immunohistochemical analysis of paraffin-embedded Mouse liver carcinoma tissue labeling BRD3 with ab325539 at 1/100 (5.12 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse liver carcinoma. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Immunohistochemical analysis of paraffin-embedded Mouse lung carcinoma tissue labeling BRD3 with ab325539 at 1/100 (5.12 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse lung carcinoma. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling BRD3 with ab325539 at 1/100 (5.12 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse colon. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

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

Lab

ChIP-sequencing - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Chromatin was prepared from MEF cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 5 x 10^6 cells and 4 µg of ab325539 [EPR30249-10]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Lab

ChIP-sequencing - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Chromatin was prepared from MEF cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 5 x 10^6 cells and 4 µg of ab325539 [EPR30249-10]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• ChIP-seq

Lab

ChIP-sequencing - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Chromatin was prepared from MEF cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 5 x 10^6 cells and 4 µg of ab325539 [EPR30249-10]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads. The Input control is also shown.

• WB

Lab

Western blot - Anti-BRD3 antibody [EPR30249-10] (AB325539)

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

Performed under reducing conditions.

In Western blot, ab325539 was shown to bind specifically to BRD3. Target of interest was observed at 100 kDa in wild-type HEK-293 cell lysates (lane 1) with no signal observed at this size in BRD3 knockout cell line (lane 2) (lane 2, knockout cell line ab266793 / knockout cell lysate ab258335).

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-BRD3 antibody [EPR30249-10] (ab325539) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

Western blot - Human BRD3 knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-brd3-knockout-hek-293t-cell-lysate-ab258335'>ab258335</a>) 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: 100 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-BRD3 antibody [EPR30249-10] (AB325539)

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

In lanes 1-2 on the right, the lysates was stored at -80degC prior to Western Blotting. The bands beneath the target band (100 kDa) are likely to be degradation products. In lanes 1-2 on the left, to minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-BRD3 antibody [EPR30249-10] (ab325539) at 1/1000 dilution

Lane 1:

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

Lane 2:

MEF (mouse embryo fibroblast) 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: 100 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-BRD3 antibody [EPR30249-10] (AB325539)

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

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

All lanes:

Western blot - Anti-BRD3 antibody [EPR30249-10] (ab325539) at 1/1000 dilution

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse liver tissue lysate at 20 µg

Lane 5:

Mouse testis tissue lysate at 20 µg

Lane 6:

Mouse spleen 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

Observed band size: 100 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-BRD3 antibody [EPR30249-10] (AB325539)

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

To minimize protein degradation, cells/tissues were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-BRD3 antibody [EPR30249-10] (ab325539) at 1/1000 dilution

Lane 1:

Neuro-2a (mouse neuroblastoma neuroblast) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

Neuro-2a transfected with siRNA specifically targeting BRD3 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: 100 kDa,36 kDa

false

Exposure time: 180s

• I-ELISA

Lab

Indirect ELISA - Anti-BRD3 antibody [EPR30249-10] (AB325539)

Indirect ELISA analysis of ab325539 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution dilution.

Antigen : Human BRD3 protein; Human BRD2 protein; Human BRD4 protein; Human BRDT protein.

Antigen concentration : 1000 ng/ml

This antibody does not cross-react with recombinant human BRD2, BRD4 or BRDT by ELISA.