Biolegend, 320202, Purified anti-human FOXP3 Antibody, 100μg

FOXP3 is a 50-55 kD transcription factor, also known as Forkhead box protein P3, Scurfin, JM2, or IPEX. It is proposed to be a master regulatory gene and more specific marker of T regulatory cells than most cell surface markers (such as CD4 and CD25). Transduced expression of FOXP3 in CD4 + /CD25 - cells has been shown to induce GITR, CD103, and CTLA4 and impart a T regulatory cell phenotype. FOXP3 is mutated in X-linked autoimmunity-allergic dysregulation syndrome (XLAAD or IPEX) in humans and in "scurfy" mice. Overexpression of FOXP3 has been shown to lead to a hypoactive immune state suggesting that this transcriptional factor is a central regulator of T cell activity. In human, unlike in mouse, two isoforms of FOXP3 have been reported: one (FOXP3) corresponding to the canonical full-length sequence; the other (FOXP3 δ2) lacking exon 2. The 259D antibody recognizes human FOXP3 epitope in the region of amino acids 105-235.
100μg
Verified Reactivity: Human
Reported Reactivity: Cynomolgus, Rhesus, Baboon, Chimpanzee
Antibody Type: Monoclonal
Host Species: Mouse
Immunogen: Full-length FOXP3 protein
Formulation: This antibody is provided in phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide.
Preparation: The antibody was purified by affinity chromatography.
Concentration: 0.5 mg/ml
Storage & Handling: The antibody solution should be stored undiluted between 2°C and 8°C.
Application: WB - Quality tested IHC-P - Verified ICFC - Reported in the literature, not verified in house SB - Community verified
Recommended Usage: Each lot of this antibody is quality control tested by immunofluorescent intracellular staining with flow cytometric analysis. For flow cytometric staining, the suggested use of this reagent is ≤ 0.5 µg per 106 cells in 100 µL volume. For Western blotting, the suggested working dilution(s) is ≤ 5.0 µg/mL in antibody dilution buffer. It is recommended that the reagent be titrated for optimal performance for each application.
Application Notes: Additional reported applications (for the relevant formats) include: Western blotting1, and immunohistochemical staining1 of acetone-fixed frozen sections and formalin-fixed paraffin-embedded sections. The 259D antibody gives strong positivity on paraffin and frozen sections and the antibody stains some epithelial cells. The binding of 206D to FOXP3 can be partially blocked by 259D, but 206D does not show significant blocking effect on 259D binding. NOTE: For flow cytometric staining with this clone, True-Nuclear™ Transcription Factor Buffer Set (Cat. No. 424401) offers improved staining and is highly recommended.
Additional Product Notes: For the use of this antibody in spatial biology applications, we have partnered with Lunaphore Technologies for demonstration of our antibodies on the COMET™. The COMET™ platform is an automated, end-to-end spatial biology solution developed for rapid and flexible multiplex tissue profiling. More information on the COMET™ and a complete list of our antibodies that have been demonstrated on the COMET™ can be found here.
Application References(PubMed link indicates BioLegend citation): Roncador G, et al. 2005 Eur. J. Immunol. 35:1681. Yang ZZ, et al. 2006. Blood 107:3639. PubMed Gavin MA, et al. 2006. P. Natl. Acad. Sci. USA 103:6659. PubMed Groh V, et al. 2006. Nature Immunology 7:755. PubMed Tran DQ, et al. 2007. Blood doi:10.1182/blood-2007-06-094656.PubMEd Long SA, et al. 2008. J Autoimmun. 30:293. PubMed Gong G, et al. 2009. Blood 113:837. PubMed Long SA, et al. 2009. Eur J. Immunol. 39:612. PubMed Long SA, et al. 2010. Diabetes. 59:407. PubMed Ferraro A, et al. 2014. PNAS. 111:1111. PubMed Vudattu NK, et al. 2014. J Immunol. 193:587. PubMed Dupont G, et al. 2014. Cytokine. 69:146. PubMed
Product Citations: Zhang S, et al. 2023. Front Cell Infect Microbiol. 13:1165790. PubMed Passerini L, et al. 2008. Int Immunol. 1.125694444. PubMed Wang R, et al. 2009. Proc Natl Acad Sci U S A. 106:13439. PubMed Humblet-Baron S, et al. 2007. J Clin Invest. 117:407. PubMed Karandikar V 2008. Blood. 111:463. PubMed Cheng B, et al. 2022. Cancer Commun (Lond). 42:17. PubMed Kaffes I, et al. 2019. Oncoimmunology. 8:e1655360. PubMed Kegler A, et al. 2019. Oncoimmunology. 8:e1621676. PubMed Pan C, et al. 2021. Oncoimmunology. 10:1883890. PubMed
RRID: AB_430884 (BioLegend Cat. No. 320201) AB_430885 (BioLegend Cat. No. 320202)
Structure: Forkhead/winged-helix transcription factor family, approximately 50 kD, contains zinc finger and forkhead domains
Distribution: Nuclear; expressed in T regulatory cells
Function: Transcription factor proposed to be a master regulatory gene in T regulatory cell development and a critical factor for immune homeostasis
Interaction: Interacts with DNA
Cell Type: Tregs
Biology Area: Cell Biology, Immunology, Transcription Factors
Molecular Family: Nuclear Markers
Antigen References: 1. Hori S, et al. 2003. Science 299:1057.
Regulation: FOXP3 is present at high levels in T regulatory cell can also be induced by T cell activation
Gene ID: 50943
UniProt: View information about FOXP3 on UniProt.org
Clone: 259D
Regulatory Status: RUO
Other Names: Forkhead box protein P3, Scurfin, JM2, IPEX, Zinc finger protein JM2
Isotype: Mouse IgG1, κ
Q: Can I stain whole blood with anti-FOXP3 using your Foxp3 staining kit?
A: It is not recommended. It is best to use PBMCs for this testing.
Q: Can FOXP3 be costained with cytokines?
A: The larger holes created by the nuclear permeabilization required for FOXP3 may allow cytokines to leak out of the cell, making it harder to detect lowly-expressed cytokines. You may have to use a control where the cells are only permeabilized through the cell membrane.
Q: If an antibody clone has been previously successfully used in IBEX in one fluorescent format, will other antibody formats work as well?
A: It’s likely that other fluorophore conjugates to the same antibody clone will also be compatible with IBEX using the same sample fixation procedure. Ultimately a directly conjugated antibody’s utility in fluorescent imaging and IBEX may be specific to the sample and microscope being used in the experiment. Some antibody clone conjugates may perform better than others due to performance differences in non-specific binding, fluorophore brightness, and other biochemical properties unique to that conjugate.
Q: Will antibodies my lab is already using for fluorescent or chromogenic IHC work in IBEX?
A: Fundamentally, IBEX as a technique that works much in the same way as single antibody panels or single marker IF/IHC. If you’re already successfully using an antibody clone on a sample of interest, it is likely that clone will have utility in IBEX. It is expected some optimization and testing of different antibody fluorophore conjugates will be required to find a suitable format; however, legacy microscopy techniques like chromogenic IHC on fixed or frozen tissue is an excellent place to start looking for useful antibodies.
Q: Are other fluorophores compatible with IBEX?
A: Over 18 fluorescent formats have been screened for use in IBEX, however, it is likely that other fluorophores are able to be rapidly bleached in IBEX. If a fluorophore format is already suitable for your imaging platform it can be tested for compatibility in IBEX.
Q: The same antibody works in one tissue type but not another. What is happening?
A: Differences in tissue properties may impact both the ability of an antibody to bind its target specifically and impact the ability of a specific fluorophore conjugate to overcome the background fluorescent signal in a given tissue. Secondary stains, as well as testing multiple fluorescent conjugates of the same clone, may help to troubleshoot challenging targets or tissues. Using a reference control tissue may also give confidence in the specificity of your staining.
Q: How can I be sure the staining I’m seeing in my tissue is real?
A: In general, best practices for validating an antibody in traditional chromogenic or fluorescent IHC are applicable to IBEX. Please reference the Nature Methods review on antibody based multiplexed imaging for resources on validating antibodies for IBEX.