Overview

  • Product name
  • Description
    Rabbit polyclonal to Brd4
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, IP, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Horse, Dog
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1100 - 1200 of Human Brd4.

    Read Abcam's proprietary immunogen policy (Peptide available as ab85811.)

  • Positive control
    • This antibody gave a positive signal in Human, Mouse and Rat Liver Tissue Lysates.
  • General notes

    "Note: Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help."

Properties

Applications

Our Abpromise guarantee covers the use of ab75898 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
ICC/IF Use a concentration of 5 µg/ml.
IP Use at 1 µg/mg of lysate.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 152 kDa (predicted molecular weight: 152 kDa).

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

  • Lane 1: Wild type HAP1 whole cell lysate (20 µg)
    Lane 2: Brd4 knockout HAP1 whole cell lysate (20 µg)
    Lane 3: HeLa whole cell lysate (20 µg)
    Lane 4: Hu brain whole cell lysate (20 µg)
    Lanes 1 - 4: Merged signal (red and green). Green - ab75898 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

  • ab75898 at 1000µg immunoprecipitating Brd4 in HEK293 whole cell lysate. Sample was incubated with primary antibody (50mM TRIS dilution buffer) and protein G for 16 hours at 4°C. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

    See Abreview

  • All lanes : Anti-Brd4 antibody (ab75898) at 1 µg/ml

    Lane 1 : Liver (Mouse) Tissue Lysate
    Lane 2 : Liver (Rat) Tissue Lysate
    Lane 3 : Human liver tissue lysate - total protein (ab29889)

    Lysates/proteins at 10 µg/ml per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP)

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 152 kDa
    Observed band size: 171 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 70 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 150 seconds
  • ab75898 (1/500) staining Brd4 in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilized with 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

    See Abreview

  • ICC/IF image of ab75898 stained MCF7 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab75898, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa, Hek293 and HepG2 cells at 5µg/ml.

References

This product has been referenced in:
  • Sui S  et al. Ferritinophagy is required for the induction of ferroptosis by the bromodomain protein BRD4 inhibitor (+)-JQ1 in cancer cells. Cell Death Dis 10:331 (2019). Read more (PubMed: 30988278) »
  • Wang C  et al. miR-204 enhances p27 mRNA stability by targeting Brd4 in head and neck squamous cell carcinoma. Oncol Lett 16:4179-4184 (2018). Read more (PubMed: 30250532) »
See all 20 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Abreviews
Application
ChIP
Sample
Mouse Cell lysate - other (JB 6P+ cell)
Negative control
IgG
Specification
JB 6P+ cell
Detection step
Real-time PCR
Type
Cross-linking (X-ChIP)
Duration of cross-linking step: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde
Positive control
DNA samples

Abcam user community

Verified customer

Submitted May 22 2017

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

Abcam user community

Verified customer

Submitted May 08 2014

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

Abcam user community

Verified customer

Submitted Jan 29 2013

Question

Product code: 75898
Lot number: GR54498-1

Inquiry: 1. Order details: - Batch number: GR54498-1 - Abcam product code: ab75898 - Purchase order number: 1040660 - Antibody storage conditions (temperature/reconstitution etc): Aliquot, then stored at -20℃ 2. Please describe the problem (high background, wrong band size, more bands, no band etc). No band 3. On what material are you testing the antibody in WB? - Species: Human - What’s cell line or tissue: HeLa - Cell extract or Nuclear extract: Cell extract - Purified protein or Recombinant protein: purified protein 3. The lysate - How much protein was loaded: 60 μg, 30ug - What lysis buffer was used: Passive lysis buffer, 5X (Promega○R, dual luciferase kit) - What protease inhibitors were used: In passive lysis buffer mentioned above, detail recipe unknown. - What loading buffer was used: 1x SDS-PAGE loading buffer - Phosphatase inhibitors: In passive lysis buffer mentioned above, detail recipe unknown. - Did you heat the samples: temperature and time: 95 ℃, 5 min 4. Electrophoresis/Gel conditions/ Transfer conditions - Reducing or non reducing gel: reducing gel - Reducing agent:SDS - Gel percentage :10% - Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, semi-dry transfer 1h and 15 min, 150 kDa protein marker nearly transferred to PVDF membrane 5. Blocking conditions - Buffer: TBST (0.1 %) - Blocking agent: milk, BSA, serum, what percentage:6 % skim milk - Incubation time: Overnight - Incubation temperature: 4℃ 6. Primary Antibody - Species:rabbit - Reacts against: Brd4 - At what dilution(s) have you tested this antibody:1:1000 (60ug),1:100 (30ug) - What dilution buffer was used: 6 % skim milk/0.1 % TBST - Incubation time: 1 hr - Incubation temperature: 25℃ - What washing steps were done: 4 times washed by 20 ml TBST, each time cost 15 min 7. Secondary Antibody - Species:donkey - Reacts against: rabbit IgG - At what dilution(s) have you tested this antibody: 1:5000 - Incubation time: 1 hr - Wash steps: 4 times washed by 20 ml TBST, each time cost 15 min - Fluorochrome or enzyme conjugate: HRP - Do you know whether the problems you are experiencing come from the secondary? No. I think that my problem I am experiencing is not come from secondary antibody, because another protein, HEXIM1, which sharing the same secondary antibody, is fine. 8. Detection method ECl, ECl+, other detection method: ECL 9. Did you apply positive and negative controls along with the samples? Please specify. No 10. Optimization attempts - How many times have you tried the Western? 4 times - Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): There is no background around 150 kDa. - Do you obtain the same results every time e.g. are background bands always in the same place? Yes. There are always no band. - What steps have you altered? No.

Read More
Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab75898.

When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. For unknown reasons some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : https://www.abcam.com/gapdh-antibody-hrp-loading-control-ab9385.html#GAPDH-Primary-antibodies-ab9385-3.jpg (or use the following: https://www.abcam.com/ab9385).BSA should then also be used in the dilutionbuffer of the antibodies instead of milk.

I would also suggest reducing the level of blocking agent in the antibody diluents to 0.1% initially to see if you can observe a signal. In order to prevent the over washing of the membrane I would also suggest reducing the wash steps to 3x5 minutes after each incubation.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Specification
HeLa
Fixative
Paraformaldehyde
Permeabilization
Yes - 0.5% Triton-X100 in PBS

Dr. Kirk Mcmanus

Verified customer

Submitted Feb 03 2012

Answer

Thank you for your inquiry. I am very sorry to hear that you are having problem with this antibody. As I understand from your e-mail, there is some problem with co-IP. Unfortunately, as the datasheet indicates this product has been tested in ICC/IF, WB applications only - but not in IP yet. This means that we do not have any IP data to share with our customers. Nevertheless, if you wish me to look through the details of the protocol, I could certainly do it for you in case some small alterations could be implemented to improve the signal. Please could you complete the questionnaire attached to this e-mail. Regarding the epitope sequence - ab75898: anti-Brd4 antibody is a polyclonal antibody. Polyclonal antibodies consist of a mixed population of IgGs each of which will recognize a different epitope. I hope this information is helpful to you. Please do not hesitate to contact us if you need any additional information or advice.

Read More

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com

Sign up