Overview

  • Product name
    Anti-FOXP3 antibody [236A/E7]
    See all FOXP3 primary antibodies
  • Description
    Mouse monoclonal [236A/E7] to FOXP3
  • Host species
    Mouse
  • Specificity
    The epitope recognized by this monoclonal antibody is between amino acids 105-236. The antibody is expected to detect full length FOXP3 as well as both cleaved forms.
  • Tested applications
    Suitable for: IHC-P, IHC-Fr, WB, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Human, Cynomolgus monkey, Rhesus monkey
  • Immunogen

    FOXP3 fusion protein.

  • Positive control
    • WB: Human thymus and tonsil lysates; HEK-293T overexpressing Human FOXP3 cell lysate. ICC/IF: Head and neck squamous cell carcinoma cells IHC-P: Human tonsil, colon and breast cancer tissue; monkey spleen tissue.
  • General notes

    This FOXP3 antibody was tested in flow cytometry. FOXP3 molecular weight is 50-55 kDa. FOXP3 antibody (ab20034) is expected to detect full length FOXP3 as well as both cleaved forms.

Properties

Applications

Our Abpromise guarantee covers the use of ab20034 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
IHC-P Use a concentration of 10 µg/ml. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.
IHC-Fr Use a concentration of 10 µg/ml.
WB Use a concentration of 4 - 5 µg/ml. Detects a band of approximately 50 kDa (predicted molecular weight: 47 kDa).
ICC/IF Use at an assay dependent concentration.
Flow Cyt Use 2µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

Target

  • Function
    Probable transcription factor. Plays a critical role in the control of immune response.
  • Involvement in disease
    Defects in FOXP3 are the cause of immunodeficiency polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) [MIM:304790]; also known as X-linked autoimmunity-immunodeficiency syndrome. IPEX is characterized by neonatal onset insulin-dependent diabetes mellitus, infections, secretory diarrhea, trombocytopenia, anemia and eczema. It is usually lethal in infancy.
  • Sequence similarities
    Contains 1 C2H2-type zinc finger.
    Contains 1 fork-head DNA-binding domain.
  • Cellular localization
    Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • AIID antibody
    • DIETER antibody
    • Forkhead box P3 antibody
    • Forkhead box protein P3 antibody
    • FOXP3 antibody
    • FOXP3_HUMAN antibody
    • FOXP3delta7 antibody
    • Immune dysregulation polyendocrinopathy enteropathy X linked antibody
    • Immunodeficiency polyendocrinopathy enteropathy X linked antibody
    • IPEX antibody
    • JM2 antibody
    • MGC141961 antibody
    • MGC141963 antibody
    • OTTHUMP00000025832 antibody
    • OTTHUMP00000025833 antibody
    • OTTHUMP00000226737 antibody
    • PIDX antibody
    • Scurfin antibody
    • XPID antibody
    see all

Images

  • ab20034 used on paraffin embedded human tonsil sections.
    Double staining: CD30 in red, FOXP3 in black, blue is haemotoxylin counterstaining of nuclei.

    As expected FOXP3 stains a sub-population of T cells.

  • Immunohistochemical analysis of human large and locally advanced breast cancers staining FOXP3 using ab20034. (a) Low level of FOXP3+, CTLA-4Treg infiltration (b) High level of FOXP3+ and CTLA-4Treg infiltration. (Itu: intratumoral Str: stromal)

  • All lanes : Anti-FOXP3 antibody [236A/E7] (ab20034) at 5 µg/ml

    Lane 1 : HEK293T cell lysate
    Lane 2 : HEk293T cell lysate overexpressing Human FOXP3
    Lane 3 : Human tonsil tissue lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 47 kDa



    ab20034 detects Human FOXP3 protein at ~50 kDa in HEK293T cells overexpressing the protein. It also detects FOXP3 in Human tonsil tissue lysate, however this band is significantly weaker in endogenous conditions. Upon higher exposure, weak bands can also be observed in HEK293T cell lysate.

    This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with Anti-FOXP3 antibody [236A/E7] (ab20034; 5 microgram per mL) overnight at 4°C. Antibody binding was detected using infrared labelled goat anti-mouse (green; 1:10000) for 1 hour at room temperature before imaging.

  • ab20034 staining FOXP3 in Cynomolgus Monkey Spleen tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% donkey serum for 20 minutes at room temperature; antigen retrieval was by heat mediation using EDTA, pH9.0. Samples were incubated with primary antibody (1/100) for 30 minutes. A Biotin-conjugated Donkey anti-mouse polyclonal (1/2000) was used as the secondary antibody.

    See Abreview

  • FoxP3+ cells mainly accumulate centrally.

    The formalin-fixed, paraffin-embedded blocks were cut into approximately <2 µm thick slices and mounted on SuperFrost Plus microscope slides (Menzel Gläser, Braunschweig, Germany). After deparaffinization and rehydration, sections were immersed into Dako Target Retrieval solution (Dako North America Inc., Carpinteria, USA), pH 6, 1/10, incubated at 97°C–99°C at 750 Watt for 2x 15 minutes, and allowed to cool to room temperature for 20 minutes. Endogenous peroxidase activity was blocked by 10-minute incubation in 7.5% hydrogen-peroxide solution (Hydroxen Peroxide Solution, Sigma Aldrich Co., Munich, Germany). Immunohistochemical staining for FoxP3 (1/180 dilution; for 60 min) was performed according to standard procedure using MACH-3 mouse alkalic phosphatase polymer detection kit from Biocare Medical Systems (Concord, USA). The slides were incubated with monoclonal mouse antibody. Chromogen Red (Dako North Amerika Inc., Carpinteria, USA) was used as chromogen for FoxP3 staining, and lastly hematoxylin counterstaining was done (Vector Laboratories, Burlingam, USA).

  • Anti-FOXP3 antibody [236A/E7] (ab20034) at 5 µg/ml + Human thymus at 20 µg

    Secondary
    IR-labelled goat anti-mouse (green; 800) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 47 kDa
    Observed band size: 50 kDa
    why is the actual band size different from the predicted?



    This blot was produced using a 4-12% Bis-Tris gel under the MOPS buffer system. The gel was run at 200V for 60 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with Anti-FOXP3 antibody [236A/E7] (ab20034; 5 microgram per mL), overnight at 4°C. Antibody binding was detected using infrared labelled goat anti-mouse (green; 1:10000) for 1 hour at room temperature before imaging.

  • Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized head and neck squamous cell carcinoma cells stained for FOXP3 (green) using ab20034 at 1/100 dilution in ICC/IF.

    See Abreview

  • ab20034 used on paraffin embedded human tonsil sections.
    FOXP3 staining is black, blue is haemotoxylin counterstaining of nuclei.

    As expected FOXP3 stains a sub-population of T cells.

  • IHC image of Foxp3 staining in Human normal tonsil formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ 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 ab20034, 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.

    *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

  • ab20034 staining FOXP3 in Human breast cancer tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with acetone/methanol (1:2), permeabilized with TBS-TritonX-100 and blocked with 10% serum for 10 minutes at 20°C. Samples were incubated with primary antibody (1/50 in PBT (PBS/BSA/Tween)) for 30 minutes at 20°C. An undiluted HRP-conjugated rat anti-mouse/rabbit IgG polyclonal was used as the secondary antibody.

    See Abreview

  • ab20034 staining FOXP3 in human colon tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent formaldehyde fixation before heat mediated antigen retrieval and then blocking with 10% serum for 2 hours at 21°C. The primary antibody was diluted 1/50 and incubated with sample for 2 hours at 21°C. An Alexa Fluor®488-conjugated rabbit polyclonal to mouse IgG was used undiluted as secondary antibody.

    See Abreview

References

This product has been referenced in:
  • Shi C  et al. CD4+ CD25+ regulatory T cells promote hepatocellular carcinoma invasion via TGF-ß1-induced epithelial-mesenchymal transition. Onco Targets Ther 12:279-289 (2019). Read more (PubMed: 30643426) »
  • Wen YH  et al. Stromal interleukin-33 promotes regulatory T cell-mediated immunosuppression in head and neck squamous cell carcinoma and correlates with poor prognosis. Cancer Immunol Immunother 68:221-232 (2019). Read more (PubMed: 30357458) »
See all 237 Publications for this product

Customer reviews and Q&As

1-8 of 8 Q&A

Question
Answer

Unfortunately, the exact epitope recognized by this Ab has not yet been mapped. However, this Ab was generated against a recombinant fragment representing Isoform 1 of Human FOXP3, amino acids aa107-196. This immunizing sequence is conserved and present among all four currently reported isoforms of Human FOXP3.

Additional information regarding this protein, its processing and isoforms can be found via the UniProt Database: UniProt Accession Q9BZS1.

Read More

Answer

Product Anti-FOXP3 antibody [236A/E7] (ab20034) is a mouse monoclonal Ab that was generated against a recombinant fragment representing Isoform 1 of Human FOXP3, amino acids aa107-196. This immunizing sequence is conserved and present among all four currently reported isoforms of Human FOXP3. As such, ab20034 will be able to detect all four isoforms of this target, provided they are present in the specific sample tested.

Additional information regarding this protein, its processing and isoforms can be found via the UniProt Database: UniProt Accession Q9BZS1.

Read More

Answer

For Mouse-on-Mouse (MOM) staining, we can recommend the following IHC detection kit: Mouse on Mouse Polymer IHC Kit (ab127055).

Alternatively, our suggested MOM Staining Protocol can be found here: MOM Staining Protocol.

Read More

Question
Answer

Unfortunately, we have not yet tested reactivity of product Anti-FOXP3 antibody [236A/E7] (ab20034) against rabbit samples. As such, use of this Ab with rabbit samples is not yet covered by our Abpromise Guarantee.

However, should you wish to test this Ab in an as-yet un-tested species or application, it may qualify for our Abtrial Testing Program.

Read More

Answer

For use of product Anti-FOXP3 antibody [236A/E7] (ab20034) in IHC-P applications, we recommend a Heat Induced Epitope Retrieval (HIER) method, using Sodium Citrate Buffer at pH 6.0.

Additional information on this method, as well as alternate AR protocols, can be found here: IHC Antigen Retrieval Protocols.

Read More

Question
Answer

Unfortunately, a directly-conjugated format of clone 236A/E7 is not currently part of our catalogue. However, we do offer several different conjugation kits for the conjugation of primary Abs to a variety of fluorophores or enzymes, should you wish to directly conjugate product Anti-FOXP3 antibody [236A/E7] (ab20034) yourself.

Additional information regarding the conjugation process, as well as a full list of our available conjugation kits, can be found here: Antibody Conjugation Kit Information.

Likewise, a BSA and Azide free format of clone 236A/E7 can be found here:

Anti-FOXP3 antibody [236A/E7] - BSA and Azide free (ab96048).

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Answer

The FOXP3 target protein is currently reported to have at least four different isoforms. The canonical FOXP3 sequence (Isoform 1) is composed of 431 amino acids, and has a calculated MW of approximately 47.2 kDa. Isoforms 2 and 4 are slightly shorter in sequence and lower in MW, whereas Isoform 3 is slightly longer and has a higher calculated MW. FOXP3 is also cleaved both near the N-terminus and C-terminus of the sequence.

Additional information regarding this protein, its processing and isoforms can be found via the UniProt Database: UniProt Accession Q9BZS1.

Read More

Answer

We have not yet tested clone 236A/E7 in Flow Cytometry/FACS applications in our own labs. However, the following publication does note use of clone 236A/E7 in Flow Cytometry/FACS:

Analysis of FOXP3 protein expression in human CD4+CD25+ regulatory T cells at the single-cell level. PMID: 15902688.

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