Validated using a knockout cell line
Recombinant
RabMAb

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

Overview

  • Product name
    Anti-Brd4 antibody [EPR5150(2)]
    See all Brd4 primary antibodies
  • Description
    Rabbit monoclonal [EPR5150(2)] to Brd4
  • Tested applications
    Suitable for: WB, IP, IHC-P, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) corresponding to Human Brd4 aa 150-250.
    Database link: O60885

  • Positive control
    • HeLa, Caco2, T.T, RAW264.7 and NIH3T3 cell lysates; HepG2 cells, Human colon carcinoma tissue and Human brain. FC: SW480 cells
  • General notes

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

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
WB 1/200 - 1/1000. Detects a band of approximately 152 kDa (predicted molecular weight: 152 kDa).

 

IP Use at an assay dependent concentration.

For unpurified use at 1ug/ml of lysate

IHC-P 1/200. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

For unpurified use at 1/40

See protocols (link: http://www.abcam.com/protocols/ihc-antigen-retrieval-protocol).

Flow Cyt 1/50.
ICC/IF 1/200.

For unpurified use at 1/40

Target

  • 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
  • Database links
  • Alternative names
    • Brd4 antibody
    • BRD4-NUT FUSION antibody
    • BRD4-NUT fusion oncoprotein antibody
    • BRD4_HUMAN antibody
    • Bromodomain containing 4 antibody
    • bromodomain containing protein 4 antibody
    • Bromodomain-containing protein 4 antibody
    • CAP antibody
    • chromosome associated protein antibody
    • HUNK1 antibody
    • HUNKI antibody
    • MCAP antibody
    • Mitotic chromosome-associated protein antibody
    • Protein HUNK1 antibody
    see all

Images



  • Predicted band size : 152 kDa

    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.

  • Unpurified ab128874 (1/500) staining Brd4 in Hela cells (green). Cells were fixed in paraformaldehyde, permeabilised in 0.5% Triton X100/PBS and counter stained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

    See Abreview

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

  • Flow cytometry analysis of SW480 (Human colorectal adenocarcinoma epithelial cell) cells labeling Brd4 (red) with purified ab128874 at a 1/50 dilution (10ug/mL). Cells were fixed with 80% methanol and permeabilized with 0.1% Tween-20. A goat anti rabbit IgG (Alexa Fluor®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).

  •  ab128874 at 1000µg immunoprecipitating Brd4 in human HEK293 whole cell lysate. Sample was incubated with unpurified primary antibody (50mM TRIS dilution buffer) and protein G for 16 hours at 4°C. For western blotting, ab131366 was used as the secondary antibody at 1/10000 dilution.

    See Abreview

  • Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution (Purified) + NIH/3T3 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
  • Immunocytochemistry/Immunofluorescence analysis of HepG2 (Human liver cell line) cells labelling Brd4 with purified ab128874 at 1/100 (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 counter stain(B).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human brain tissue labelling Brd4 with purified ab128874 at 1/200. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. An undiluted Goat Anti-Rabbit IgG H&L (HRP)  was used as the secondary antibody at. Counter stained with Hematoxylin.

  • Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/1000 dilution (purified) + HeLa 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

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

  • Anti-Brd4 antibody [EPR5150(2)] (ab128874) at 1/200 dilution (unpurified) + HeLa 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 kDa

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

  • Equilibrium disassociation constant (KD)
    Learn more about KD

    Click here to learn more about KD

References

This product has been referenced in:
  • Hogg SJ  et al. BET-Bromodomain Inhibitors Engage the Host Immune System and Regulate Expression of the Immune Checkpoint Ligand PD-L1. Cell Rep 18:2162-2174 (2017). ChIP ; Human . Read more (PubMed: 28249162) »
  • Wei W  et al. Class I histone deacetylases are major histone decrotonylases: evidence for critical and broad function of histone crotonylation in transcription. Cell Res 27:898-915 (2017). ChIP ; Human . Read more (PubMed: 28497810) »

See all 14 Publications for this product

Customer reviews and Q&As

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Testis)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Ventana CC1 (EDTA pH8.5)
Permeabilization
No
Specification
Testis
Blocking step
Ventana blocking agent as blocking agent for 4 minute(s) · Concentration: 100% · Temperature: 36°C
Fixative
Formaldehyde
Username

Abcam user community

Verified customer

Submitted Jun 20 2017

Application
Western blot
Loading amount
30 µg
Gel Running Conditions
Non-reduced Non-Denaturing (Native)
Sample
Mouse Cell lysate - whole cell (pancreatic cancer)
Specification
pancreatic cancer
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Ms. Jamie Plevy

Verified customer

Submitted Sep 26 2014

Application
Immunoprecipitation
Immuno-precipitation step
Protein G
Sample
Human Cell lysate - whole cell (HEK293)
Specification
HEK293
Total protein in input
1000 µg
Username

Abcam user community

Verified customer

Submitted Apr 25 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (mouse embryonic fibroblasts)
Loading amount
15 µg
Specification
mouse embryonic fibroblasts
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jan 23 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (pancreatic tumor)
Loading amount
25 µg
Specification
pancreatic tumor
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Nov 07 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (pancreatic cancer cell line)
Loading amount
25 µg
Specification
pancreatic cancer cell line
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Nov 07 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% TritonX100 in PBS
Username

Dr. Kirk Mcmanus

Verified customer

Submitted Oct 19 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (endometrial adenocarcinoma)
Specification
endometrial adenocarcinoma
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate
Permeabilization
Yes - PBST
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Oct 08 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (endometrial adenocarcinoma)
Specification
endometrial adenocarcinoma
Fixative
Paraformaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrate
Permeabilization
Yes - PBST
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Oct 08 2012

Thank you very much for your call last week and for your patience while I have been in touch with the lab regarding ab128874.

The immunogen is a peptide taken from between amino acids 175 and 200 of human Brd4.

I hope this informati...

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