Name: Anti-Brd4 antibody [EPR5150(2)]
SKU: ab128874
Rating: 4 (17 reviews)

Anti-Brd4 antibody [EPR5150(2)] is a rabbit monoclonal antibody used for detecting Brd4 in western blotting (WB), immunohistochemistry on paraffin-embedded sections (IHC-P), immunocytochemistry/immunofluorescence (ICC/IF), and flow cytometry. Suitable for human and mouse samples.

- Cited in over 160 publications
- Affinity purified using Protein A
- Recombinant format ensures unrivaled batch-to-batch consistency; no need for same-lot requests
- Specificity confirmed with BRD4 knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation

Images

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

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	IHC-P	WB	ICC/IF	Flow Cyt (Intra)
Human	Tested	Tested	Tested	Tested
Mouse	Expected	Tested	Expected	Expected
Rat	Predicted	Predicted	Predicted	Predicted

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/200	Notes For unpurified use at 1/40 Perform heat-mediated antigen retrieval before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Predicted
Predicted

Species	Dilution info	Notes
Species Rat	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/200 - 1/1000	Notes -
Species Human	Dilution info 1/200 - 1/1000	Notes -

Predicted
Predicted

Species	Dilution info	Notes
Species Rat	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/200	Notes For unpurified use at 1/40

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Predicted
Predicted

Species	Dilution info	Notes
Species Rat	Dilution info -	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/50	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info Use at an assay dependent concentration.	Notes -

Predicted
Predicted

Species	Dilution info	Notes
Species Rat	Dilution info -	Notes -

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Target data

See full target information BRD4

Function

Chromatin reader protein that recognizes and binds acetylated histones and plays a key role in transmission of epigenetic memory across cell divisions and transcription regulation (PubMed:20871596, PubMed:23086925, PubMed:23317504, PubMed:29176719, PubMed:29379197). Remains associated with acetylated chromatin throughout the entire cell cycle and provides epigenetic memory for postmitotic G1 gene transcription by preserving acetylated chromatin status and maintaining high-order chromatin structure (PubMed:22334664, PubMed:23317504, PubMed:23589332). During interphase, plays a key role in regulating the transcription of signal-inducible genes by associating with the P-TEFb complex and recruiting it to promoters (PubMed:16109376, PubMed:16109377, PubMed:19596240, PubMed:23589332, PubMed:24360279). Also recruits P-TEFb complex to distal enhancers, so called anti-pause enhancers in collaboration with JMJD6 (PubMed:16109376, PubMed:16109377, PubMed:19596240, PubMed:23589332, PubMed:24360279). BRD4 and JMJD6 are required to form the transcriptionally active P-TEFb complex by displacing negative regulators such as HEXIM1 and 7SKsnRNA complex from P-TEFb, thereby transforming it into an active form that can then phosphorylate the C-terminal domain (CTD) of RNA polymerase II (PubMed:16109376, PubMed:16109377, PubMed:19596240, PubMed:23589332, PubMed:24360279). Regulates differentiation of naive CD4(+) T-cells into T-helper Th17 by promoting recruitment of P-TEFb to promoters (By similarity). Promotes phosphorylation of 'Ser-2' of the C-terminal domain (CTD) of RNA polymerase II (PubMed:23086925). According to a report, directly acts as an atypical protein kinase and mediates phosphorylation of 'Ser-2' of the C-terminal domain (CTD) of RNA polymerase II; these data however need additional evidences in vivo (PubMed:22509028). In addition to acetylated histones, also recognizes and binds acetylated RELA, leading to further recruitment of the P-TEFb complex and subsequent activation of NF-kappa-B (PubMed:19103749). Also acts as a regulator of p53/TP53-mediated transcription: following phosphorylation by CK2, recruited to p53/TP53 specific target promoters (PubMed:23317504). Isoform B. Acts as a chromatin insulator in the DNA damage response pathway. Inhibits DNA damage response signaling by recruiting the condensin-2 complex to acetylated histones, leading to chromatin structure remodeling, insulating the region from DNA damage response by limiting spreading of histone H2AX/H2A.x phosphorylation.

Alternative names

HUNK1, BRD4, Bromodomain-containing protein 4, Protein HUNK1

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR5150(2)
Purification technique: Affinity purification Protein A
Dissociation constant: 3.2 x 10^-11 M
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

Anti-Brd4 antibody [EPR5150(2)] (ab128874) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in Flow Cyt (Intra), ICC/IF, IHC-P and WB.

Anti-Brd4 antibody [EPR5150(2)] (ab128874) was first used in a scientific publication in 2014 and has been cited over 163 times in peer reviewed journals. It's performance in Western Blot in human samples is trusted by the scientific community.

Abcam's high quality manufacturing and validation processes ensure Anti-Brd4 antibody [EPR5150(2)] (ab128874) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.

The specificity of Anti-Brd4 antibody [EPR5150(2)] (ab128874) has been confirmed by Western Blot testing in Brd4 knockout Hap1 cells.

Anti-Brd4 antibody [EPR5150(2)] (ab128874) has 17 independent reviews from customers.

Anti-Brd4 antibody [EPR5150(2)] (ab128874) specifically detects Brd4 (UniProt ID: O60885; Molecular weight: 153kDa) and is sold in 100 µL and 1 mL selling sizes.

Conjugation-ready, carrier free format available for antibody clone EPR5150(2) - Anti-Brd4 antibody [EPR5150(2)] - BSA and Azide free ab182446.

Antibody clone EPR5150(2) is also available pre-conjugated to a variety of labels for your convenience - Alexa Fluor^® 488, Alexa Fluor^® 647, HRP, Alexa Fluor^® 594 (Alexa Fluor® 488 Anti-Brd4 antibody [EPR5150(2)] ab197606, Alexa Fluor® 647 Anti-Brd4 antibody [EPR5150(2)] ab197608, HRP Anti-Brd4 antibody [EPR5150(2)] ab197609, Alexa Fluor® 594 Anti-Brd4 antibody [EPR5150(2)] ab207057).

Top cited antibody for this target with >200 citations. A highly specific BRD4 antibody, essential for studying bromodomain-containing protein 4, epigenetic regulation and chromatin remodeling. This antibody is crucial in cancer research, particularly in understanding BRD4's role in transcription regulation, chemoresistance and tumor progression and is widely used in studies of BRD4 inhibitors and BET proteins. Research has shown that BRD4 (and BET family proteins) may be promising therapeutic targets for various Myc-driven cancers, such as Burkitt’s lymphoma and certain acute myeloid leukaemia's and is studied is a potential therapeutic target.

Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

BRD4 also known as Bromodomain-containing protein 4 is a member of the BET (bromodomain and extra-terminal domain) family. This protein has a molecular weight of approximately 120 - 150 kDa often used in western blot analysis to identify its presence. It binds to acetylated lysines on histone proteins facilitating the regulation of gene expression. BRD4 is mainly expressed in the nucleus and plays an essential role in remodeling chromatin structure therefore influencing transcription processes.

Biological function summary

This protein influences various cellular activities by regulating transcriptional elongation of specific genes. BRD4 forms part of a complex with P-TEFb (Positive Transcription Elongation Factor b) which phosphorylates the C-terminal domain of RNA polymerase II facilitating transcription through chromatin. It binds to acetylated histones and non-histone proteins linking it to active transcription sites and contributing to the dynamic regulation of gene expression in response to cellular signals.

Pathways

BRD4 participates in key signaling networks like the NF-κB pathway and MYC signaling. It interacts with transcription factors and other proteins such as Cyclin T1 and CDK9 within the P-TEFb complex to regulate gene expression. BRD4 also collaborates with MYC a critical transcription factor regulating cell growth and proliferation to modulate pathways related to cellular growth and survival.

Associated diseases and disorders

BRD4 has been linked to cancer and inflammation-related conditions. Overexpression or mutations in BRD4 can lead to aberrant cell proliferation in certain cancers including breast and leukemia where it influences the expression of oncogenes. In inflammation BRD4 modulates the expression of pro-inflammatory cytokines. Through these pathways it is connected to related proteins like NF-κB which orchestrates cellular responses to inflammation and stress signals.

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14 product images

Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Lanes 1 - 2: Merged signal (red and green). Green - ab128874 observed at 220 kDa. Red - loading control Mouse anti Vinculin observed at 125 kDa.
ab128874 was shown to react with Brd4 in wild-type cells in Western blot with loss of signal observed in BRD4 knockout sample. Wild-type and BRD4 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab128874 and Mouse anti Vinculin overnight at 4 °C at a 1 in 200 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/200 dilution
Lane 1: Wild-type HAP1 cell lysate at 20 µg
Lane 2: BRD4 knockout HAP1 cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 152 kDa
Observed band size: 220 kDa
Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Lane 1: Wild-type HAP1 cell lysate (20 μg)
Lane 2: Brd4 knockout HAP1 cell lysate (20 μg)
Lane 3: HeLa cell lysate (20 μg)

Lanes 1 - 4: Merged signal (red and green). Green - ab128874 observed at 150 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
ab128874 was shown to recognize Brd4 when Brd4 knockout samples were used, along with additional cross-reactive bands. Wild-type and Brd4 knockout samples were subjected to SDS-PAGE. ab128874 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were diluted at 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
Lane 1: Western blot - Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Lanes 1 - 3: Western blot - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) at 1/10000 dilution
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Brd4 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Secondary
All lanes: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/10000 dilution
Predicted band size: 152 kDa, 36 kDa
Ren et al PLoS Pathog. 2016 Oct; 12(10): e1005950. Published online 2016 Oct 20. doi: 10.1371/journal.ppat.1005950
Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Brd4 immunofluorescence staining of HeLa cells using rabbit anti-Brd4 antibody

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
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Brd4 immunohistochemistry staining of colon carcinoma using rabbit anti-Brd4 antibody

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.
Flow Cytometry (Intracellular) - Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Brd4 flow cytometry staining of SW480 cells using rabbit anti-Brd4 antibody

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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (Black) (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730). Blue (unlabeled control) - Cell without incubation with primary antibody and secondary antibody (Blue).
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
Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Brd4 immunofluorescence staining of HepG2 cells using rabbit anti-Brd4 antibody

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).
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
Image courtesy of a customer review submitted by Dr. Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada
Immunocytochemistry/ Immunofluorescence - Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Brd4 immunofluorescence staining of HeLa cells using rabbit anti-Brd4 antibody

Unpurified ab128874 (1/500 dilution) staining Brd4 in HeLa (Human epithelial cell line 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.
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
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
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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Brd4 immunohistochemistry staining of human colon using rabbit anti-Brd4 antibody

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.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Brd4 immunohistochemistry staining of human colon using rabbit anti-Brd4 antibody

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.