Overview

  • Product name
  • Description
    Rabbit polyclonal to FOXO3A
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Dog
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 650 to the C-terminus of Human FOXO3A.

    Read Abcam's proprietary immunogen policy (Peptide available as ab24031.)

  • Positive control
    • Recombinant Human FOXO3A protein (ab114191) can be used as a positive control in WB. This antibody gave a positive signal in the following whole cell lysates: Jurkat (Human T cell lymphoblast-like cell line) This antibody gave a positive signal in the following tissue lysates: Mouse Heart Mouse Skeletal Muscle Rat Heart This antibody gave a positive result in IHC in the following FFPE tissue: Human normal lung.

Properties

Applications

Our Abpromise guarantee covers the use of ab23683 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 Use a concentration of 1 µg/ml. Detects a band of approximately 90 kDa (predicted molecular weight: 71 kDa).
IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF Use a concentration of 5 µg/ml.

Target

  • Function
    Transcriptional activator which triggers apoptosis in the absence of survival factors, including neuronal cell death upon oxidative stress. Recognizes and binds to the DNA sequence 5'-[AG]TAAA[TC]A-3'.
  • Tissue specificity
    Ubiquitous.
  • Involvement in disease
    Note=A chromosomal aberration involving FOXO3 is found in secondary acute leukemias. Translocation t(6;11)(q21;q23) with MLL/HRX.
  • Sequence similarities
    Contains 1 fork-head DNA-binding domain.
  • Post-translational
    modifications
    In the presence of survival factors such as IGF-1, phosphorylated on Thr-32 and Ser-253 by AKT1/PKB. This phosphorylated form then interacts with 14-3-3 proteins and is retained in the cytoplasm. Survival factor withdrawal induces dephosphorylation and promotes translocation to the nucleus where the dephosphorylated protein induces transcription of target genes and triggers apoptosis. Although AKT1/PKB doesn't appear to phosphorylate Ser-315 directly, it may activate other kinases that trigger phosphorylation at this residue. Phosphorylated by STK4 on Ser-209 upon oxidative stress, which leads to dissociation from YWHAB/14-3-3-beta and nuclear translocation. Phosphorylated by PIM1.
  • Cellular localization
    Cytoplasm > cytosol. Nucleus. Translocates to the nucleus upon oxidative stress and in the absence of survival factors.
  • Information by UniProt
  • Database links
  • Alternative names
    • AF6q21 antibody
    • AF6q21 protein antibody
    • DKFZp781A0677 antibody
    • FKHR2 antibody
    • FKHRL 1 antibody
    • FKHRL1 antibody
    • FKHRL1P2 antibody
    • Forkhead (Drosophila) homolog (rhabdomyosarcoma) like 1 antibody
    • Forkhead box O3 antibody
    • Forkhead box O3A antibody
    • Forkhead box protein O3 antibody
    • Forkhead box protein O3A antibody
    • Forkhead Drosophila homolog of in rhabdomyosarcoma like 1 antibody
    • Forkhead homolog (rhabdomyosarcoma) like 1 antibody
    • Forkhead in rhabdomyosarcoma like 1 antibody
    • Forkhead in rhabdomyosarcoma-like 1 antibody
    • FOX O3A antibody
    • FOXO2 antibody
    • foxo3 antibody
    • FOXO3_HUMAN antibody
    • FOXO3A antibody
    • MGC12739 antibody
    • MGC31925 antibody
    see all

Images

  • All lanes : Anti-FOXO3A antibody (ab23683) at 1 µg/ml

    Lane 1 : Jurkat whole cell lysate (ab7899)
    Lane 2 : Jurkat whole cell lysate (ab7899) with Human FOXO3A peptide (ab24031) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 71 kDa
    Observed band size: 90 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 50 kDa (possible cleavage fragment), 50 kDa (possible cross reactivity)



    ab23683 detects a band at approximately 90 kDa that corresponds in size to that seen for FOXO3A. This protein has been shown to migrate at a size larger than the predicted molecular weight (see Yin et al., J. Biol. Chem., Vol. 279, Issue 44, 45721-45727). It also detects a band at 50 kDa which could be a cleavage fragment. Both bands are partially blocked by the addition of the immunizing peptide.
  • All lanes : Anti-FOXO3A antibody (ab23683) at 1 µg/ml

    Lane 1 : Heart (Mouse) Tissue Lysate - normal tissue
    Lane 2 : Mouse skeletal muscle tissue lysate - total protein (ab29711)
    Lane 3 : Heart (Rat) Tissue Lysate - normal tissue

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Predicted band size: 71 kDa
    Observed band size: 90 kDa why is the actual band size different from the predicted?
    Additional bands at: 16 kDa, 27 kDa. We are unsure as to the identity of these extra bands.



    ab23683 detects a band at approximately 90 kDa that corresponds in size to that seen for FOXO3A. This protein has been shown to migrate at a size larger than the predicted molecular weight (see Yin et al., J. Biol. Chem., Vol. 279, Issue 44, 45721-45727).

  • ICC/IF image of ab23683 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 (ab23683, 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% HepG2 fixed (10 min) HepG2 cells at 5µg/ml.
  • IHC image of FOXO3A staining in Human normal lung formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab23683, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

References

This product has been referenced in:
  • Gao Y  et al. MicroRNA-155 increases colon cancer chemoresistance to cisplatin by targeting forkhead box O3. Oncol Lett 15:4781-4788 (2018). Read more (PubMed: 29552117) »
  • Huang J  et al. MiR-155 is upregulated in patients with active tuberculosis and inhibits apoptosis of monocytes by targeting FOXO3. Mol Med Rep 12:7102-8 (2015). Read more (PubMed: 26324048) »
See all 2 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Question
Answer

Thank you for confirming these details and for your cooperation.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number ######.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Answer

Thank you for your message and for providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a free of charge replacement vial as requested.

Would you like to try another vial of ab23683? Or was there another antibody you would like to try, what is the product number?

I look forward to hearing from you with details of how you would like to proceed.

Read More

Answer

Thank you for providing this further information.

I appreciate your cooperation. I have managed to trace your order, which is within the guarantee period. Reviewing the details:

1. The band at 50 kDa could possibly be a cleavage product, and there is as you say a band at this size on the blot shown on the datasheet. I have conducted a literature search for more information, and regrettably am not able to confirm this. However, you may like to consider trying a further search.

2. I can suggest to try a different molecular weight marker, and also some fresh samples to reduce risk of degradation.

I will be pleased to provide a free of charge replacement or credit note if the antibody is not working as expectedwith the suggestions provided.

Thank you for your time. I look forward to hearing from you with the results from the next experiment and hope we can resolve this case as soon as possible.

Read More

Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this batch of antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab23683 is tested and covered by our 6 month guarantee for use inWB and in mouse, rat andhuman samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimize the results. I would also appreciate if you can confirm some further details:

1. Please provide the order number and date of purchase. How has the antibody been stored?

2. Could you confirm if the same type of samples have been used with this batch of the antibody? What species and cell line or tissue type have been tested?

3. Have the samples been reduced and denatured? This will help to ensure the are in the correct conformation to run at the correct molecular weight.

4. How have samples been stored? I can suggest to try fresh samples, with protease inhibitors added to the lysis buffer.

5. If the band is changing as expected on cell treatment, then this indicates the bandcould be the correct protein. I can suggest to try a different molecular weight marker to check the accuracy?

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

Read More

Answer

Thank you for your reply. I do not believe there will be any problems with the antibody being in a different tube but please let me know if the customer has issues with the antibody being packaged different. I will keep you posted when I hear anything.  

Read More

Answer

I am unsure as to why ab23683 was shipped in a 1ml vial instead of a 0.5ml vial. I am in the process of trying to get an answer for you. I was wandering though if you feel the quality of the product is being affected by it being shipped in a different container? I will inform you as soon as I have an answer to your original enquiry.

Read More
Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Colon)
Specification
Colon
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Dako Target Retrieval Solution
Permeabilization
No
Blocking step
5 minutes of peroxidase block then 10 minutes of protein block. These are ready-to-use reagents purchased from Dako as blocking agent for 15 minute(s) · Concentration: 100% · Temperature: 20°C

Mr. Antibody Solutions

Verified customer

Submitted Dec 16 2010

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Human Cell lysate - whole cell (Jurkat)
Total protein in input
400 µg
Specification
Jurkat
Immuno-precipitation step
Protein A

Mr. Antibody Solutions

Verified customer

Submitted Apr 28 2008

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

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