Overview

  • Product name
    Anti-c-Fos (phospho T325) antibody
    See all c-Fos primary antibodies
  • Description
    Rabbit polyclonal to c-Fos (phospho T325)
  • Host species
    Rabbit
  • Specificity
    ab27793 recognises the cFos phosphorilated at threonine 325 form.
  • Tested applications
    Suitable for: ICC/IF, ChIP, WB, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic phosphopeptide derived from the region of human cFos that contains threonine 325.

  • Positive control
    • Human epidermoid carcinoma (A431) cells stimulated with EGF or 15% serum.

Properties

Applications

Our Abpromise guarantee covers the use of ab27793 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 2 - 3 µg/ml.
ChIP Use 1-3µg for 106 cells.
WB 1/1000. Predicted molecular weight: 41 kDa.
IP Use at an assay dependent concentration. PubMed: 20410304

Target

  • Function
    Nuclear phosphoprotein which forms a tight but non-covalently linked complex with the JUN/AP-1 transcription factor. In the heterodimer, FOS and JUN/AP-1 basic regions each seems to interact with symmetrical DNA half sites. On TGF-beta activation, forms a multimeric SMAD3/SMAD4/JUN/FOS complex at the AP1/SMAD-binding site to regulate TGF-beta-mediated signaling. Has a critical function in regulating the Has a critical function in regulating the development of cells destined to form and maintain the skeleton. It is thought to have an important role in signal transduction, cell proliferation and differentiation.
  • Sequence similarities
    Belongs to the bZIP family. Fos subfamily.
    Contains 1 bZIP domain.
  • Post-translational
    modifications
    Phosphorylated in the C-terminal upon stimulation by nerve growth factor (NGF) and epidermal growth factor (EGF). Phosphorylated, in vitro, by MAPK and RSK1. Phosphorylation on both Ser-362 and Ser-374 by MAPK1/2 and RSK1/2 leads to protein stabilization with phosphorylation on Ser-374 being the major site for protein stabilization on NGF stimulation. Phosphorylation on Ser-362 and Ser-374 primes further phosphorylations on Thr-325 and Thr-331 through promoting docking of MAPK to the DEF domain. Phosphorylation on Thr-232, induced by HA-RAS, activates the transcriptional activity and antagonizes sumoylation. Phosphorylation on Ser-362 by RSK2 in osteoblasts contributes to osteoblast transformation.
    Constitutively sumoylated by SUMO1, SUMO2 and SUMO3. Desumoylated by SENP2. Sumoylation requires heterodimerization with JUN and is enhanced by mitogen stimulation. Sumoylation inhibits the AP-1 transcriptional activity and is, itself, inhibited by Ras-activated phosphorylation on Thr-232.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Activator protein 1 antibody
    • AP 1 antibody
    • C FOS antibody
    • Cellular oncogene c fos antibody
    • Cellular oncogene fos antibody
    • FBJ murine osteosarcoma viral (v fos) oncogene homolog (oncogene FOS) antibody
    • FBJ murine osteosarcoma viral oncogene homolog antibody
    • FBJ murine osteosarcoma viral v fos oncogene homolog antibody
    • FBJ Osteosarcoma Virus antibody
    • FOS antibody
    • FOS protein antibody
    • FOS_HUMAN antibody
    • G0 G1 switch regulatory protein 7 antibody
    • G0/G1 switch regulatory protein 7 antibody
    • G0S7 antibody
    • Oncogene FOS antibody
    • p55 antibody
    • proto oncogene c Fos antibody
    • Proto oncogene protein c fos antibody
    • Proto-oncogene c-Fos antibody
    • v fos FBJ murine osteosarcoma viral oncogene homolog antibody
    see all

Images

  • All lanes : Anti-c-Fos (phospho T325) antibody (ab27793) at 1/1000 dilution (diluted in a 3% Milk TBST buffer.)

    Lane 1 : Non EGF treated A431 cell lysate
    Lane 2 : EGF treated A431 cell lysate
    Lane 3 : EGF treated A431 cell lysate treated with Lambda phosphatase

    Secondary
    All lanes : goat F(ab’)2 anti rabbit IgG HRP conjugate

    Predicted band size: 41 kDa
    Observed band size: 58 kDa
    why is the actual band size different from the predicted?



    The figure shows that the phosphorylation of cFos on threonine 325 is induced by EGF treatment and that Lambda phosphatase treatment eliminates the signal, thereby demonstrating the phospho specificity of ab27793.
  • Immunofluorescence analysis of c-Fos (phospho T325) was done on 70% confluent log phase HeLa cell treated with 200 nM of PMA for 20 minutes. The cell were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phosc-Fos (phospho T325)Rabbit Polyclonal Antibody (ab27793) at 2 ug/ml in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate at a dilution of 1/2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with DAPI. F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin. Panel d is a merged image showing nuclear and cytoplasmic localization. Panel e is untreated cell with no signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

  • Chromatin Immunoprecipitation (ChIP) was performed using Anti-c-Fos (phospho T325) antibody (ab27793) 3 ug on sheared chromatin from 2 million A431 cells treated with EGF (200ng/ml), for 30 minutes. Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system with optimized PCR primer pairs for the promoter of active IL-6, CDKN1A gene, used as positive control target, and the SAT2, used as negative control target. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

References

This product has been referenced in:
  • Lu JY  et al. Estrogen enhances mismatch repair by induction of MLH1 expression via estrogen receptor-ß. Oncotarget 8:38767-38779 (2017). Read more (PubMed: 28404976) »
  • Nakakuki T  et al. Ligand-specific c-Fos expression emerges from the spatiotemporal control of ErbB network dynamics. Cell 141:884-96 (2010). WB ; Human . Read more (PubMed: 20493519) »
See all 4 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Application
Western blot
Sample
Chicken Cell lysate - whole cell (Chicken thrombocyte)
Gel Running Conditions
Reduced Denaturing
Loading amount
10 µg
Specification
Chicken thrombocyte
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Mrs. Katie Elliott

Verified customer

Submitted Apr 25 2017

Question
Answer

Thank you for contacting us.

I am sorry that this antibody did not perform as stated on the datasheet, I have asked our Finance department to issue a credit note for you. The credit note may be used in one of the following ways:

(1) Redeemed against the original invoice if this hasn't already been paid.
(2) Held on the account for use against a future order.
(3) A full refund can be offered where no other invoices are outstanding.

Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge.

To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website.

The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB.

I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

Read More

Answer

Thank you for your reply.


I am sorry to hear that my suggestions did not improve your results with this antibody.I am happy to arrange for a refund. Would you please provide your order number for this product?


I look forward to your reply so that I may assist you further. Please do not hesitate to contact us if you have any additional questions.

Read More

Answer

Thank you for contacting Abcam regarding ab27793.


I am sorry that you have been experiencing difficulties with this antibody in WB. I have reviewed the protocol information you provided and would like to make some suggestions to improve your results.


When using a phospho-specific antibody it is highly advised against using milk as a blocking reagent or diluent. I would recommend switching to 5% BSA in PBST instead. Also switching to TBST often decreases background. I would suggest the following:


How much sample are you loading? I would recommend loading 50-75ug total protein in each lane. Then, block for at least 1 hr at room temperature in 5% BSA in TBST. Incubate with the primary antibody (also diluted in 5% BSA in TBST) for 1 hour at room temperature rather than overnight. Test 1:1000, 1:2000, and 1:4000. Perform washes with TBST. Incubate the secondary antibody diluted in 5% BSA (dilute according to manufacturers instructions). It may be necessary to titrate the secondary antibody concentration as well. Wash and develop as you normally would.


I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions or if these suggestions do not improve your results with this antibody and I will be happy to provide a replacement or refund per our Abpromise guarantee.

Read More

Answer

Thank you for your enquiry. Ab27793 has not yet been tested for application in IF (only Western blotting so far)but ab17933 - Rabbit polyclonal to c-Fos (phospho T232) - has been tested in IF. There is an image on the online datasheet with more more information. Please contact us again if you have any additional questions.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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