Overview

  • Product name
    Anti-c-Jun antibody - ChIP Grade
    See all c-Jun primary antibodies
  • Description
    Rabbit polyclonal to c-Jun - ChIP Grade
  • Host species
    Rabbit
  • Specificity
    Antibody detects endogenous levels of c-Jun protein around Serine 243.
  • Tested applications
    Suitable for: ChIP, ICC/IF, IHC-P, IP, WB, ELISA, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, African green monkey
    Predicted to work with: Chicken, Cow, Pig
  • Immunogen

    Synthetic peptide within Human c-Jun aa 210-259. The exact sequence is proprietary.
    Sequence:

    HLPQQMPVQHPRLQALKEEPQTVPEMPGETPPLSPIDMESQERIKAERKR


    Database link: P05412

  • Positive control
    • ChIP: Human endothelial cells (EA.hy926). IHC-P: Human breast carcinoma tissue. ICC/IF: HepG2 cells. WB: Extracts of HeLa cells. Recombinant Human c-Jun protein. Flow Cytometry: Jurkat cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab31419 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
ChIP Use at an assay dependent concentration.
ICC/IF Use a concentration of 1 µg/ml.
IHC-P 1/50 - 1/100.
IP Use at an assay dependent concentration.
WB 1/500 - 1/1000. Predicted molecular weight: 36 kDa.
ELISA 1/20000.
Flow Cyt 1/300.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Transcription factor that recognizes and binds to the enhancer heptamer motif 5'-TGA[CG]TCA-3'. Promotes activity of NR5A1 when phosphorylated by HIPK3 leading to increased steroidogenic gene expression upon cAMP signaling pathway stimulation. Involved in activated KRAS-mediated transcriptional activation of USP28 in colorectal cancer (CRC) cells (PubMed:24623306). Binds to the USP28 promoter in colorectal cancer (CRC) cells (PubMed:24623306).
  • Sequence similarities
    Belongs to the bZIP family. Jun subfamily.
    Contains 1 bZIP (basic-leucine zipper) domain.
  • Post-translational
    modifications
    Ubiquitinated by the SCF(FBXW7), leading to its degradation. Ubiquitination takes place following phosphorylation, that promotes interaction with FBXW7.
    Phosphorylated by CaMK4 and PRKDC; phosphorylation enhances the transcriptional activity. Phosphorylated by HIPK3. Phosphorylated by DYRK2 at Ser-243; this primes the protein for subsequent phosphorylation by GSK3B at Thr-239. Phosphorylated at Thr-239, Ser-243 and Ser-249 by GSK3B; phosphorylation reduces its ability to bind DNA. Phosphorylated by PAK2 at Thr-2, Thr-8, Thr-89, Thr-93 and Thr-286 thereby promoting JUN-mediated cell proliferation and transformation. Phosphorylated by PLK3 following hypoxia or UV irradiation, leading to increase DNA-binding activity.
    Acetylated at Lys-271 by EP300.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Activator protein 1 antibody
    • AP 1 antibody
    • AP1 antibody
    • cJun antibody
    • Enhancer Binding Protein AP1 antibody
    • Jun Activation Domain Binding Protein antibody
    • JUN antibody
    • Jun oncogene antibody
    • JUN protein antibody
    • Jun proto oncogene antibody
    • JUN_HUMAN antibody
    • JUNC antibody
    • Oncogene JUN antibody
    • p39 antibody
    • Proto oncogene c jun antibody
    • Proto oncogene cJun antibody
    • Proto-oncogene c-jun antibody
    • Transcription Factor AP 1 antibody
    • Transcription factor AP-1 antibody
    • Transcription Factor AP1 antibody
    • V jun avian sarcoma virus 17 oncogene homolog antibody
    • V jun sarcoma virus 17 oncogene homolog (avian) antibody
    • V jun sarcoma virus 17 oncogene homolog antibody
    • V-jun avian sarcoma virus 17 oncogene homolog antibody
    • vJun Avian Sarcoma Virus 17 Oncogene Homolog antibody
    see all

Images

  • ChIP analysis using ab31419 binding c-Jun in human endothelial cells (EA.hy926). Cells were cross-linked for 10 minutes with formaldehyde then incubated with undiluted primary antibody for 10 hours at 4°C in 1x ChIP buffer. Protein binding was detected using real-time PCR.
    Positive control: Position 89340150-89340297 in chromosome 11 (has a validated c-Jun site).
    Negative Control: Igr5 intron 3 (contains no c-Jun binding site).

    See Abreview

  • Paraffin-embedded human breast carcinoma tissue stained for c-Jun with ab31419 at a 1/50 dilution in immunohistochemical analysis.

    Left panel: Untreated.

    Right panel: Pre-incubated with synthesized peptide.

  • ICC/IF image of  HepG2 (Human liver hepatocellular carcinoma cell line) cells labeling c-Jun (green) with ab31419 at 1 µg/ml. The cells were fixed in 4% PFA (10 minutes) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with ab31419 at 1 µg/ml overnight at +4 °C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) ab150077 used at a 1/1000 dilution for 1 hour. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

  • All lanes : Anti-c-Jun antibody - ChIP Grade (ab31419) at 1/500 dilution

    Lane 1 : Extracts of Hela (Human epithelial cell line from cervix adenocarcinoma) cells
    Lane 2 : Extracts of Hela (Human epithelial cell line from cervix adenocarcinoma) cells with immunizing peptide

    Predicted band size: 36 kDa
    Observed band size: 43 kDa
    why is the actual band size different from the predicted?

  • c-Jun was immunoprecipitated from HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate using ab31419 at a 1/500 dilution.

    Lane 1: Control IgG IP in HEK-293T whole cell lysate.

    Lane 2: ab31419 in HEK-293T whole cell lysate.

    Lane 3: c-Jun in HEK-293T whole cell lysate 500 µg (Input).

    For western blotting an HRP-conjugated swine anti-rabbit polyclonal was used as the secondary antibody.

    See Abreview

  • Overlay histogram showing Jurkat (Human T cell leukemia cell line from peripheral blood) cells stained with ab31419 (blue line) at a 1/300 dilution. The cells were prepared as follows: Spin down, wash in FACS buffer 1x, fix, wash 2x, and stain with primary antibody overnight. Buffer was 0.1% sodium azide with FBS in phosphate buffered solution. Cells were fixed using paraformaldehyde and permeabilized using Triton X-100 and NP40. Cells were gated by isolating cell population from plot of SSC-A / FSA-A. The secondary antibody used was a FITC-conjugated Goat anti-Rabbit polyclonal, diluted 1/100.

    See Abreview

  • Anti-c-Jun antibody - ChIP Grade (ab31419) at 1/500 dilution + Recombinant Human c-Jun protein (ab54318) at 0.01 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 36 kDa


    Exposure time: 30 seconds

References

This product has been referenced in:
  • Yu CY  et al. MicroRNA-125b-5p improves pancreatic ß-cell function through inhibiting JNK signaling pathway by targeting DACT1 in mice with type 2 diabetes mellitus. Life Sci N/A:N/A (2019). Read more (PubMed: 30684546) »
  • Lin B  et al. c-Jun suppresses the expression of WNT inhibitory factor 1 through transcriptional regulation and interaction with DNA methyltransferase 1 in gallbladder cancer. Mol Med Rep 17:8180-8188 (2018). Read more (PubMed: 29693707) »
See all 41 Publications for this product

Customer reviews and Q&As

1-8 of 8 Q&A

Question

Hello , Here is the complain of two antibodies .

1) Abcam catalogue no:

Ab31419, Ab47476

2) a) Abcam order number (not required):

1148646


b) Abcam product lot number:

c-Jun> GR50836-9, ATF-2> GR72060-1

3) Description of the problem (high background, wrong band size, more bands, no band etc):

No bands

4) Antibody storage conditions (temperature/reconstitution etc):

-20°C


5) a)Sample (Species):


c-Jun and ATF2 from HeLa


b) Type of sample (Cell extract/Nuclear extract/Purified protein/Recombinant protein etc).:


Whole cell extract

6) a) Sample preparation:

Lysis buffer



RIPA





Protease inhibitors



AEBSF, 2 mM
Aprotinin, 0.3 μM
Bestatin, 130 μM
EDTA, 1 mM
E-64, 14 μM
Leupeptin, 1 μM

PMSF, 20 mM





Phosphatase inhibitors



-





Reducing agent



SDS and β-ME





Boiling for ≥5 min?



5 minutes







b) Amount of protein loaded:







Protein loaded ug/lane or cells/lane



5 μg/lane







7) a) Electrophoresis/Gel conditions:







Reducing or Non Reducing gel



Reducing





Percentage of gel



15%





Volts applied



75





Time applied



3 hours

















b) Transfer or blocking conditions:







Type of membrane



PVDF





Protein transfer verified



Ponceau, β-actin





Blocking agent and concentration



3% BSA





Blocking time



30 minutes





Blocking temperature



25°C

















8) Primary antibody (If more than one was used, describe in “additional notes”):









Concentration or dilution



1/1000





Diluent buffer



1% BSA + 0.05% Tween-20 in 1X PBS





Incubation time



2 hours





Incubation temperature



25°C





Washing: Buffer Used



0.05% Tween-20 in 1X PBS





Number of washes



3







9) Secondary antibody:









Species



Goat





Reacts against



Rabbit





Concentration or dilution



1/10,000





Diluent buffer



1% BSA + 0.05% Tween-20 in 1X PBS





Incubation time



2 hours





Incubation temperature:



25°C





Fluorochrome or enzyme conjugate



HRP





Washing: Buffer Used



0.05% Tween-20 in 1X PBS





Number of washes



3







10) Detection method (ECL, ECL plus, etc.):









ECL





11) Positive and negative controls used:









Positive control



β-actin





Negative control











12) a) How many times you have run this staining?







Twice







b) Do you obtain the same results every time?







No







C) What steps have you altered to try and optimize the use of this antibody?







Reducing blocking, primary and secondary incubation times while attempting the second time. Washing was made slightly rigorous as compared to he first time. Performed a Dot blot to check for performance of secondary antibody and it works fine. Increased amount of loaded protein to 10 μg/lane.







Regards

Read More
Answer

Thank you for your enquiry regarding ab31419 and ab47476; and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with these two antibodies.
I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:
1) The loaded amount of protein seems to be a bit low: 5 μg/lane, normally we would suggest loading at least 25-30 μg/lane protein per lane.
2) Since c-Jun and ATF2 are both mainly present in the nuclei, it would be worth preparing nuclear fraction (rather than whole cell lysate) to enrich the target proteins in the sample if it is possible and apply them for WB.
3) Customer may also need to test different dilutions (1/500 or even 1/250) to make sure that the experiment is carried out under the optimal conditions. This is even more important since only 5 μg instead of 30 μg lysate was loaded.
I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact our Technical Department again.

Read More

Answer

Thank you for contacting us.

I do wish to aplogize for the delays in getting information about this product to you. I have been informded by the originator of this product that the images are using ductal breast carcinoma however as of this time we do not have any information regarding the percentages of breat carcinoma would be positive.



This product is covered by our Abpromise guarantee. We are happy provide scientific support at any time. If you are using the product in species and applications listed on the datasheet and contact us within six months of purchase, we are also happy to replace or refund the product should we not be able to help you to resolve the issue. More information on our Abpromise may be found at the following link:

https://www.abcam.com/index.html?pageconfig=abpromise

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Read More

Answer

Thank you very much for contacting us.

I just wanted to let you know that I am still working on your inquiry. I have contacted the originator of this product for the information you requested, and I am awaiting a reply.

I can provide some information regarding postive controls for this product. This antibody has been tested successfully in the following tissues/cell lines as shown in on the datasheet and within publicatiobns referenced:

Hela, Jurkat, Mouse small bowel adenoma, U2OS, 3T3, COS7, PC12 , Primary Prostate Epithelia [PMID: 19592505], dorsal mouse skin [PMID: 19762425]


I am very sorry for the delay. I will forward to you the information as soon as I receive it. Thank you for your patience.

Read More

Answer

Thank you for your reply.

Unfortunately we do not have another C-Jun antibody that is tested in ChIP. Please let me know how you would like to proceed. We do not currently stock a C-Fos antibody that will work in ChIP but if you would like to test an antibody then please let me know. You may be eligible for a testing discount.

I look forward to your reply.

Read More

Question
Answer

Thank you for your reply and with clarifying information.

We have not received any other complaints about this antibody, so I am sorry to hear you have been experiencing problems. It is troubling that it is not working in both ChIP and WB. I would like to offer you a replacement vial of the same or an alternative product, or a refund/credit. Please could you confirm your preference and also provide the following details:

- Date of order/delivery
- Primary antibody dilution

I look forward to your reply.

Read More

Answer

Thank you for contacting us.

I am extremely sorry for the delay in investigating this case. The quality of our products is very important to us, so I am sorry to hear this antibody is not giving satisfactory results. I have reviewed the information provided and have some comments:

1. You detail that a positive control antibody has not been used. I would encourage you to include an antibody against another protein or histone modification that is localised to the regions being analyzed by PCR. This would confirm whether the problem is isolated to just the c-jun antibody or the ChIP procedure.

2. Is the antibody working succesfully in other procedures?

3. X-ChIP is more appropriate for chromatin associated proteins, so I would continue with this technique to ensure that that c-jun remains associated to the chromatin.

4. Which beads were used for the immunoprecipitation? I would suggest protein A or Protein G beads, protein A have a higher affinity for rabbit antibodies.

5. Is the Jun protein over expressed in the fibroblasts, or are you analyzing endogenous protein?

6. Could you confirm whether the Nucleobond DNA purification kit is suitable for small DNA fragments? A PCR based purification kit is suitable for ChIP as the DNA fragments will also be of a similar size.

I look forward to your reply.

Read More

Question

Thanks for your answer. I answered the questions in your e-mail (see below). Although I have to say that I can not answer most of the questions since they are thought for western blot and I am doing ChIP.
Just let me know if you have any suggestions or you can offer me another antibody which works better for ChIP.
Thanks a lot.

Montse

Order Details
Antibody code: ab31419

Problem
Choose: No signal or weak signal

Lot number GR50836-2

Purchase order number
or preferably Abcam order number: ######
General Information
Antibody storage conditions (temperature/reconstitution etc): stored at –20oC

Description of the problem (high background, wrong band size, more bands, no
band etc.)
I tried different ChIP protocols (with and without corsslink) and I get always really low signals in the qPCR and no specif enreachment in regions where I know that Jun is binding.

Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant
protein etc.) I´ve used human fibroblasts induced and uninduced with TPA

Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)

Amount of protein loaded

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel
etc.)

Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Washstep)

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time,
Wash step)

Detection method (ECL, ECLPlus etc.)

Positive and negative controls used (please specify)

Optimization attempts (problem solving)
How many times have you tried the Western?

Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?

What steps have you altered?

Additional Notes:

Read More
Answer

Thank you for your message and for kindly completing the ChIP questionnaire.

Reviewing your comments, I agree it would be better for us to review the ChIP procedure. I am sorry I sent the WB questionnaire as you mentioned you had also been using this antibody is WB unsucessfully too, and thought it may be better to start with this.

With regards to other c-Jun antibodies for use in ChIP, I am sorry we do not have any others in our catalog that have been tested and guaranteed in this application. I would like to reassure you that we monitor feedback closely on a weekly basis, and we are not currently concerned about the general quality of this antibody or batch. However, it is possible that you have received a bad vial and I can confirm this antibody is covered by our guarantee for ChIP and human samples.

In this case, I would be pleased to investigate further, and am forwarding the ChIP questionnaire below. I would appreciate if you could take time to complete this and I hope this will be more helpful to you. We would also appreciate if you are able to provide any data that would help us to assess the results.

Thank you for your patience and cooperation. I look forward to receiving the completed questionnaire.



Order Details Antibody code
Lot number
Purchase order number or preferably Abcam order number

General Information
Antibody storage conditions (temperature/reconstitution etc)

Description of the problem (no signal, high background, low signal, not reproducible)

X-CHIP or N-CHIP? (please give details)

Sample (Species/Tissue/Cell Line/Nuclear or whole extract)

Sample preparation (Buffer/Protease inhibitors/Starting material - number of cells or protein content etc.)

Cross linking the cells(Cross linking agent/Cross link time/Optimise cross linking time?)

DNA fragmentation (Method/Optimisation/Fragment size etc.)

IP step (Matrix/Concentration/Chromatin amount/Incubation/Native IP testing/Washing conditions/Elution)

Decrosslinking (Time/Temperature/Proteinase K details)

DNA purification (Phenol, DNA extraction kit, other?)

Pellet after precipitation? (please specify)

DNA amplification (Semiquantitative PCR, Real Time PCR, Sybr green, Taqman?)

Primers tested on genomic or input DNA? (please specify)

Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

Positive and negative controls used (please specify)

Optimization attempts (problem solving) How many times have you tried the CHIP?

Do you obtain the same results every time? Yes No

Was a no-template control included in the PCR? (please specify)

What steps have you altered?

Additional Notes:


Data: We would appreciate if you are able to provide any data that would help us to assess the results.

Read More

Answer

I apologize for the delay in replying to your question. Ab8754 recognizes non-phosphorylated proteins. As for the other two antibodies, ab31417 and ab31419, here is the information I obtained from the originator: We did WB again on these two antibodies. We did find that the MWs were different. It may caused by the different phosphorylation at the different sites. These two antibodies do not recognize phospho protein. They recognize the non-phospho-domains in which the phospho sites are not phosphorylated. This may cause the MWs to be different. The peptide successfully blocked reaction in our experiments. I hope that this information helps. Please do not hesitate to contact us in the future for advice or information.

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