Recombinant
RabMAb

Recombinant Anti-B7H4 antibody [EPR20236-9] (ab269455)

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Overview

  • Product name

    Anti-B7H4 antibody [EPR20236-9]
    See all B7H4 primary antibodies

  • Description

    Rabbit monoclonal [EPR20236-9] to B7H4

  • Host species

    Rabbit

  • Tested applications

    Suitable for: ICC/IF, Flow Cyt, IP, WBmore details
    Unsuitable for: IHC-P

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Recombinant fragment within Human B7H4 aa 1 to the C-terminus. The exact sequence is proprietary.
    Database link: Q7Z7D3

  • Positive control

    • WB: SK-BR-3, T47D and Mouse uterus lysates. ICC/IF: HEK-293T cell line transfected with a HA-tagged B7-H4 expression vector. FC: 293T (human embryonic kidney epithelial cell) transfected with myc-tagged B7-H4 expression vector. IP: SK-BR-3 cells.

  • General notes

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

Applications

Our Abpromise guarantee covers the use of ab269455 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 1/500.
Flow Cyt 1/60.
IP 1/30.
WB 1/1000. Detects a band of approximately 28, 45, 65 kDa (predicted molecular weight: 31 kDa).
  • Application notes
    Is unsuitable for IHC-P.

    • Target

    • Function

      Negatively regulates T-cell-mediated immune response by inhibiting T-cell activation, proliferation, cytokine production and development of cytotoxicity. When expressed on the cell surface of tumor macrophages, plays an important role, together with regulatory T-cells (Treg), in the suppression of tumor-associated antigen-specific T-cell immunity. Involved in promoting epithelial cell transformation.

    • Tissue specificity

      Overexpressed in breast, ovarian, endometrial, renal cell (RCC) and non-small-cell lung cancers (NSCLC). Expressed on activated T- and B-cells, monocytes and dendritic cells, but not expressed in most normal tissues (at protein level). Widely expressed, including in kidney, liver, lung, ovary, placenta, spleen and testis.

    • Sequence similarities

      Belongs to the immunoglobulin superfamily. BTN/MOG family.
      Contains 2 Ig-like V-type (immunoglobulin-like) domains.

    • Post-translational
      modifications

      N-glycosylated.

    • Cellular localization

      Cell membrane. Expressed at the cell surface. A soluble form has also been detected.
    • Information by UniProt

    • Database links

      • Alternative names

        • B7 family member, H4 antibody
        • B7 H4 antibody
        • B7 homolog 4 antibody
        • B7 superfamily member 1 antibody
        • B7 superfamily, member 1 antibody
        • B7-H4 antibody
        • B7h.5 antibody
        • B7h4 antibody
        • B7S1 antibody
        • B7x antibody
        • BC032925 antibody
        • Immune costimulatory protein B7-H4 antibody
        • Immune costimulatory protein B7H4 antibody
        • MGC41287 antibody
        • PRO1291 antibody
        • Protein B7S1 antibody
        • RP11 229A19.4 antibody
        • T cell costimulatory molecule B7x antibody
        • T-cell costimulatory molecule B7x antibody
        • V set domain-containing T cell activation inhibitor 1 antibody
        • V-set domain-containing T-cell activation inhibitor 1 antibody
        • VCTN1 antibody
        • Vtcn1 antibody
        • VTCN1_HUMAN antibody
        see all

      Images

      • Western blot - Anti-B7H4 antibody [EPR20236-9] (ab269455)
        Western blot - Anti-B7H4 antibody [EPR20236-9] (ab269455)
        All lanes : Anti-B7H4 antibody [EPR20236-9] (ab269455) at 1/1000 dilution

        Lane 1 : SK-BR-3 (human breast adenocarcinoma epithelial cell), whole cell lysate
        Lane 2 : T47D (human ductal breast epithelial tumor epithelial cell), whole cell lysate
        Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
        Lane 4 : HT-29 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
        Lane 5 : Mouse uterus tissue lysate

        Lysates/proteins at 20 µg per lane.

        Secondary
        All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

        Predicted band size: 31 kDa
        Observed band size: 28,45,65 kDa
        why is the actual band size different from the predicted?



        Blocking and dilution buffer: 5% NFDM/TBST.

        Exposure time: 114 seconds.

        B7H4 is N-glycosylated protein. The level of B7H4 glycosylation varies between different cells and tissues. The bands at 65kDa and 45kDa represent glycosylated B7H4. The band at 28kDa represents non-glycosylated B7H4. (PMID: 15878339).

        Negative control: HT-29 and HeLa. (PMID: 15878339).

      • Immunoprecipitation - Anti-B7H4 antibody [EPR20236-9] (ab269455)
        Immunoprecipitation - Anti-B7H4 antibody [EPR20236-9] (ab269455)

        B7H4 was immunoprecipitated from 0.35 mg SK-BR-3 (Human breast adenocarcinoma epithelial cell) whole cell lysate with ab269455 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab269455 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

        Lane 1: SK-BR-3 whole cell lysate 10ug.

        Lane 2: ab269455 IP in SK-BR-3 whole cell lysate.

        Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab269455 in SK-BR-3 whole cell lysate.

        Blocking and dilution buffer and concentration: 5% NFDM/TBST.

        Exposure time: 30 seconds.

        B7H4 is N-glycosylated protein. The level of B7H4 glycosylation varies between different cells and tissues. The band at 65kDa represents glycosylated B7H4. The band at 28kDa represents non-glycosylated B7H4 (PMID: 15878339).

      • Flow Cytometry - Anti-B7H4 antibody [EPR20236-9] (ab269455)
        Flow Cytometry - Anti-B7H4 antibody [EPR20236-9] (ab269455)

        Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized HEK-293T (human embryonic kidney epithelial cell) transfected with myc-tagged B7-H4 expression vector cells labelling B7H4 with ab269455 at 1/60 dilution (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left). PMID: 23722540 and PMID: 27632942 showed that cell surface expression of B7-H4 hard to detected in vitro cultured cell lines, and all the commercial B7H4 antibodies that recommend FC as an application use the overexpression system. Our RabMab also could recognise overexpressed B7H4.The overexpression plasmid contains the full length aa1-282 of human B7H4(Q7Z7D3) with HA tag in pcDNA3.1(+) vector.

        Cells were surface stained with rabbit IgG (Left) or ab269455 (Right). Then fixed with 2% PFA followed by intracellularly stained with anti-HA tag conjugated to Alexa Fluor® 647.

      • Immunocytochemistry/ Immunofluorescence - Anti-B7H4 antibody [EPR20236-9] (ab269455)
        Immunocytochemistry/ Immunofluorescence - Anti-B7H4 antibody [EPR20236-9] (ab269455)

        Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human embryonic kidney epithelial cell) cells labelling B7H4 with ab269455 at 1/500 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic and membranous staining in HEK-293T cell line transfected with a HA-tagged B7-H4 expression vector. Alexa Fluor® 647 anti-HA.11 Epitope Tag Antibody was used at 200 dilution to detecte the overexpressed protein (Red). The nuclear counterstain was DAPI (Blue).

        Secondary antibody only control: Secondary antibody is ab150077 AlexaFluo®488 Goat anti-Rabbit secondary at 1/1000 dilution.

      Protocols

      To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

      Click here to view the general protocols

      References

      Publishing research using ab269455? Please let us know so that we can cite the reference in this datasheet.

      ab269455 has not yet been referenced specifically in any publications.

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