Recombinant
RabMAb

Anti-FOXO3A antibody [EP1949Y] (ab53287)

Overview

  • Product name
    Anti-FOXO3A antibody [EP1949Y]
    See all FOXO3A primary antibodies
  • Description
    Rabbit monoclonal [EP1949Y] to FOXO3A
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    corresponding to Human FOXO3A (N terminal).

  • Positive control
    • MCF7, Jurkat, SH-SY5Y and HCT 116 cell lysates and human breast and thyroid carcinoma tissue. This antibody gave a positive result when used in the following methanol fixed cell lines: HeLa
  • General notes

    Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.

    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 ab53287 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 1/1000. Detects a band of approximately 90 kDa (predicted molecular weight: 71 kDa).

For unpurified use at 1/2000 - 1/5000. 

IHC-P 1/250.

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

ICC/IF 1/50 - 1/100.

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

Anti-FOXO3A antibody [EP1949Y] images

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling FOXO3A with purified ab53287 at 1/100 dilution (6.1μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor® 488) was used as the secondary antibody at 1/1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue sections labeling FOXO3A with purified ab53287 at 1/250 dilution (2.4 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer, PH9. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used undiluted. PBS instead of the primary antibody was used as the negative control.

  • All lanes : Anti-FOXO3A antibody [EP1949Y] (ab53287) at 1/1000 dilution (purified)

    Lane 1 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
    Lane 2 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
    Lane 3 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate
    Lane 4 : HCT 116 (Human colorectal carcinoma cell line) whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size : 71 kDa
    Observed band size : 90 kDa (why is the actual band size different from the predicted?)

    Blocking and diluting buffer: 5% NFDM/TBST.

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa cells labeling FOXO3A with unpurified ab53287. The cells were 100% methanol fixed (5 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 ab53287 at 1/200 dilution overnight at +4°C. The secondary antibody (green) was goat anti-rabbit DyLight® 488 (ab96899) IgG (H+L) used at a 1/250 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.
  • Anti-FOXO3A antibody [EP1949Y] (ab53287) at 1/5000 dilution (unpurified) + Jurkat cell lysate at 10 µg

    Secondary
    Goat anti-Rabbit HRP labeled at 1/2000 dilution

    Predicted band size : 71 kDa
    Observed band size : 90 kDa (why is the actual band size different from the predicted?)
  • All lanes : Anti-FOXO3A antibody [EP1949Y] (ab53287) at 1/2000 dilution (unpurified)

    Lane 1 : HCT116 cells treated with DMSO (control) whole cell lysate
    Lane 2 : HCT116 cells treated with 2 µM SAHA treatment for 24 hours, whole cell lysate

    Lysates/proteins at 30 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 71 kDa
    Observed band size : 70 kDa (why is the actual band size different from the predicted?)


    Exposure time : 10 seconds

    This image is courtesy of an abreview submitted by Christian Marx

    Non-specific band : 130 kDa

    Blocking buffer: 5% milk

    Diluting buffer: 1% Milk in PBS-T buffer

    See Abreview

  • Immunohistochemical analysis of human breast carcinoma tissue labeling FOXO3A with unpurified ab53287 at 1/50. The sections were fixed with Formalin and embedded in paraffin. 

References for Anti-FOXO3A antibody [EP1949Y] (ab53287)

This product has been referenced in:
  • Cheung K  et al. CD31 signals confer immune privilege to the vascular endothelium. Proc Natl Acad Sci U S A 112:E5815-24 (2015). ICC/IF ; Mouse . Read more (PubMed: 26392551) »
  • Furgason JM  et al. Whole genome sequencing of glioblastoma multiforme identifies multiple structural variations involved in EGFR activation. Mutagenesis 29:341-50 (2014). Read more (PubMed: 25103728) »

See all 6 Publications for this product

Product Wall


For IHC-P, the antigen retrieval solution used was citrate buffer pH6 - heat-mediated.

Antigen Retrieval

Immerse slides into staining dish containing Antigen Retrieval Solution.

Place covered staining dish into the rice ...

Read More
Application
Western blot
Sample
Mouse Cell lysate - whole cell (MEF cell line)
Loading amount
30 µg
Specification
MEF cell line
Treatment
DMSO control and 2 µM SAHA for 24 hrs
Gel Running Conditions
Reduced Denaturing (13%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Mr. Christian Marx

Verified customer

Submitted Feb 22 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HCT116 cell line)
Loading amount
30 µg
Specification
HCT116 cell line
Treatment
DMSO control and 2 µM SAHA treatment for 24 hrs
Gel Running Conditions
Reduced Denaturing (13%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Mr. Christian Marx

Verified customer

Submitted Feb 20 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Jurkat cells)
Loading amount
200000 cells
Specification
Jurkat cells
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Nov 24 2009

Thank you for your enquiry. Please refer to the following ICC/IF protocols for this product. Solutions and reagents - PBST washing buffer: 1XPBS/0.1% Tween-20 (Dulbecco’s Phosphate Buffered Salts, 1X, catalog #21-031-CV from Mediatech, Inc.) ...

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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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