Anti-Brd4 antibody [EPR5150(2)]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling Brd4 with ab128874 at a concentration of 1 µg/ml. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. ab128874 Anti-Brd4 antibody [EPR5150(2)] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling Brd4 with ab128874 at a concentration of 3 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab128874 Anti-Brd4 antibody [EPR5150(2)] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Immunocytochemical analysis of HeLa cells showing co-localization of BRD4 using ab128874 at 1/500 dilution (red) with CDK9 (green) following infection with HSV-1. Cells were fixed with 4% paraformaldehyde (10 min at RT) and permeabilzed with 0.2% Triton X-100 (10 min). AlexaFluor^®-conjugated secondary antibodies were used at 1/1000 dilution. The nuclear counter stain is DAPI 9blue).

From Figure 5b of Ren et al.

Reproduced under the Creative Commons Licence fromRen et al PLoS Pathog. 2016 Oct; 12(10) : e1005950. Published online 2016 Oct 20. doi : 10.1371/journal.ppat.1005950 PMID : 27764245

Ren et al PLoS Pathog. 2016 Oct; 12(10): e1005950. Published online 2016 Oct 20. doi: 10.1371/journal.ppat.1005950

• ICC/IF

AbReview32451****

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Unpurified ab128874 (1/500 dilution) staining Brd4 in HeLa cells from cervix adenocarcinoma) cells (green). Cells were fixed in paraformaldehyde permeabilized in 0.5% Triton X100/PBS and counterstained with DAPI in order to highlight the nucleus (red).

For further experimental details please refer to Abreview.

Image courtesy of an abreview submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Intracellular Flow Cytometry analysis of SW480 (Human colorectal adenocarcinoma epithelial cell) cells labeling Brd4 (red) with purified ab128874 at a 1/50 dilution (10μg/mL). Cells were fixed with 80% methanol and permeabilized with 0.1% Tween-20. A goat anti rabbit IgG (Alexa Fluorr^®488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Immunocytochemistry/Immunofluorescence analysis of HepG2 (Human liver cell line) cells labeling Brd4 with purified ab128874 at 1/100 dilution (Panel A). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/200 dilution) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain(Panel B).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Unpurified ab128874 at 1/100 dilution staining Brd4 in human colon carcinoma tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Different batches of ab128874 were tested on HeLa (Human cervix adenocarcinoma epithelial cell) lysate at 0.1 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 152 kDa.

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874)

Predicted band size: 152 kDa

false

• WB

Supplier Data

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Blocking and Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/200 dilution

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg/mL

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 152 kDa

Observed band size: 152 kDa

false

• WB

Supplier Data

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Blocking and Diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 152 kDa

Observed band size: 152 kDa

false

• WB

Lab

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of 293T whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab128874 (1/1000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique.

ab128874 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected under these conditions. This data was generated as part of a structured antibody stability study supporting Abcam’s transition from cold‑chain to ambient shipping.

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution

All lanes:

293T Whole cell lysate at 15 µg with NDFM/TBST

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

false

Exposure time: 5s

• WB

Lab

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

Western blot : Anti-Brd4 antibody [EPR5150(2)] ab128874 staining at 1/1000 dilution, shown in green; Anti-Calnexin antibody [CANX/1543] ab238078 loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 200 kDa for Brd4L and 100 kDa for Brd4S in Wild-type MCF7 with a diminished signal detected in CRISPR-Cas9 Edited MCF7 cells expressing a heterozygous Brd4 knockout. Nuclear fractions exhibit enriched Brd4 expression in MCF7 and HeLa cell lines. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% NFDM 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 IgG 800CW and Goat anti-Mouse IgG 680RD at 1/20,000 dilution. Abbreviations : W – Whole cell lysate; N – Nuclear fraction lysate; WT – Wild-Type.

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution

Lane 1:

Wild type MCF7 (Whole cell lysate)

Lane 2:

Wild type MCF7 (Nuclear fraction lysate)

Lane 3:

Heterozygous MCF7 (Wild type)

Lane 4:

Heterozygous MCF (Nuclear fraction lysate)

Lane 5:

HeLa (whole cell lysate)

Lane 6:

HeLa (Nuclear fraction lysate)

Secondary

Lanes 1 - 6:

Goat anti-Rabbit IgG 800CW at 1/20000 dilution

Lanes 1 - 6:

Goat anti-Mouse IgG 680RD at 1/20000 dilution

Observed band size: 100 kDa,200 kDa

false

• WB

Supplier Data

Western blot - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

All lanes:

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution

All lanes:

NIH/3T3 (Mouse embryo fibroblast cell line) cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 152 kDa

Observed band size: 152 kDa

false

• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Brd4 antibody [EPR5150(2)] (AB128874)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.