Recombinant Anti-Brd4 antibody [EPR5150(2)] (ab128874)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR5150(2)] to Brd4
- Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Mouse, Human
Related conjugates and formulations
Overview
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Product name
Anti-Brd4 antibody [EPR5150(2)]
See all Brd4 primary antibodies -
Description
Rabbit monoclonal [EPR5150(2)] to Brd4 -
Host species
Rabbit -
Tested applications
Suitable for: Flow Cyt (Intra), WB, IHC-P, ICC/IFmore details -
Species reactivity
Reacts with: Mouse, Human
Predicted to work with: Rat -
Immunogen
Synthetic peptide within Human Brd4 aa 150-250. The exact sequence is proprietary.
Database link: O60885 -
Positive control
- WB: HeLa, Caco-2, TT, RAW 264.7 and NIH/3T3 cell lysate. Wild-type HAP1 lysate. ICC/IF: HeLa and HepG2 cells. IHC-P: Human colon carcinoma and brain tissue. Flow Cyt (intra): SW480 cells. IP: HEK-239 cell lysate.
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General notes
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 production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Dissociation constant (KD)
KD = 3.20 x 10 -11 M Learn more about KD -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR5150(2) -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab128874 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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Flow Cyt (Intra) |
1/50.
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WB | (9) |
1/200 - 1/1000. Detects a band of approximately 152 kDa (predicted molecular weight: 152 kDa).
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IHC-P | (3) |
1/200. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
For unpurified use at 1/40 |
ICC/IF | (4) |
1/200.
For unpurified use at 1/40 |
Notes |
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Flow Cyt (Intra)
1/50. |
WB
1/200 - 1/1000. Detects a band of approximately 152 kDa (predicted molecular weight: 152 kDa). |
IHC-P
1/200. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. For unpurified use at 1/40 |
ICC/IF
1/200. For unpurified use at 1/40 |
Target
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Function
Plays a role in a process governing chromosomal dynamics during mitosis. -
Tissue specificity
Ubiquitously expressed. -
Involvement in disease
Note=A chromosomal aberration involving BRD4 is found in a rare, aggressive, and lethal carcinoma arising in midline organs of young people. Translocation t(15;19)(q14;p13) with NUT which produces a BRD4-NUT fusion protein. -
Sequence similarities
Contains 2 bromo domains. -
Cellular localization
Nucleus. - Information by UniProt
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Database links
- Entrez Gene: 23476 Human
- Entrez Gene: 57261 Mouse
- Entrez Gene: 362844 Rat
- Omim: 608749 Human
- SwissProt: O60885 Human
- SwissProt: Q9ESU6 Mouse
- Unigene: 187763 Human
- Unigene: 253518 Mouse
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Alternative names
- Brd4 antibody
- BRD4-NUT FUSION antibody
- BRD4-NUT fusion oncoprotein antibody
see all
Images
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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, 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 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging. -
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 from Ren et al PLoS Pathog. 2016 Oct; 12(10): e1005950. Published online 2016 Oct 20. doi: 10.1371/journal.ppat.1005950
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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.
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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).
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All lanes : Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/200 dilution
Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : BRD4 knockout HAP1 cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 152 kDa
Observed band size: 220 kDa why is the actual band size different from the predicted?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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
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Immunocytochemistry/Immunofluorescence analysis of HepG2 (Human liver cell line) cells labeling Brd4 with purified ab128874 at 1/100 (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) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain(Panel B).
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Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution (Purified) + NIH/3T3 (Mouse embryo fibroblast cell line) cell lysate at 10 µg
Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution
Predicted band size: 152 kDa
Observed band size: 152 kDa -
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.
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Unpurified ab128874 (1/500) 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.
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Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution (purified) + HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg
Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution
Predicted band size: 152 kDa
Observed band size: 152 kDaBlocking and Diluting buffer and concentration: 5% NFDM/TBST
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Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/200 dilution (unpurified) + HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg/ml
Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution
Predicted band size: 152 kDa
Observed band size: 152 kDaBlocking and Diluting buffer and concentration: 5% NFDM/TBST
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (139)
ab128874 has been referenced in 139 publications.
- Kawaharada M et al. Novel cytological model for the identification of early oral cancer diagnostic markers: The carcinoma sequence model. Oncol Lett 23:76 (2022). PubMed: 35111245
- Irie T et al. IL-34 in hepatoblastoma cells potentially promote tumor progression via autocrine and paracrine mechanisms. Cancer Med 11:1441-1453 (2022). PubMed: 35132816
- Hua T et al. BRD4 Inhibition Attenuates Inflammatory Pain by Ameliorating NLRP3 Inflammasome-Induced Pyroptosis. Front Immunol 13:837977 (2022). PubMed: 35154163
- Lindén M et al. FET fusion oncoproteins interact with BRD4 and SWI/SNF chromatin remodelling complex subtypes in sarcoma. Mol Oncol 16:2470-2495 (2022). PubMed: 35182012
- Saglam O et al. Expression of epigenetic pathway related genes in association with PD-L1, ER/PgR and MLH1 in endometrial carcinoma. PLoS One 17:e0264014 (2022). PubMed: 35226658