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  1. Link

    cd276-antibody-epncir122-ab134161.pdf

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Immunology Immune Interventions Transplantation
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-CD276 antibody [EPNCIR122] (ab134161)

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Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
  • Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
  • Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (ab134161)
  • Immunocytochemistry/ Immunofluorescence - Anti-CD276 antibody [EPNCIR122] (ab134161)
  • Immunoprecipitation - Anti-CD276 antibody [EPNCIR122] (ab134161)
  • Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
  • Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (ab134161)
  • Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (ab134161)
  • Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
  • Immunohistochemistry (Frozen sections) - Anti-CD276 antibody [EPNCIR122] (ab134161)
  • Anti-CD276 antibody [EPNCIR122] (ab134161)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPNCIR122] to CD276
  • Suitable for: IHC-Fr, WB, IP, Flow Cyt, ICC/IF
  • Knockout validated
  • Reacts with: Mouse, Human, Recombinant fragment

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 594 Alexa Fluor® 647 Carrier Free

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Overview

  • Product name

    Anti-CD276 antibody [EPNCIR122]
    See all CD276 primary antibodies
  • Description

    Rabbit monoclonal [EPNCIR122] to CD276
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-Fr, WB, IP, Flow Cyt, ICC/IFmore details
    Unsuitable for: IHC-P
  • Species reactivity

    Reacts with: Mouse, Human, Recombinant fragment
  • Immunogen

    Recombinant full length protein corresponding to Human CD276. Alternate injections with Human recombinant protein and Human CD276 over-expressing CHO cells.
    Database link: Q5ZPR3
    (Peptide available as ab182198)

  • Positive control

    • WB: LnCaP, CHO-K1 cells lysates transfected with human and mouse CD276. Flow Cyt: HEK293 and THP1 cells. ICC/IF: HEK293 cells.
  • General notes

    This antibody was developed as part of a collaboration between Epitomics, the National Cancer Institute's Center for Cancer Research and the lab of Brad St. Croix.

    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.

    Rat: We have preliminary internal testing data to indicate this antibody may not react with this species. Please contact us for more information.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: 40% Glycerol, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPNCIR122
  • Isotype

    IgG
  • Research areas

    • Immunology
    • Immune Interventions
    • Transplantation
    • Immunology
    • Adaptive Immunity
    • T Cells
    • CD
    • Cancer
    • Tumor biomarkers
    • Other
    • Stem Cells
    • Hematopoietic Progenitors
    • Lymphoid
    • T Lymphocytic Lineage
    • Neuroscience
    • Processes

Associated products

  • Alternative Versions

    • Anti-CD276 antibody [EPNCIR122] - Low endotoxin, Azide free (ab209895)
    • Alexa Fluor® 488 Anti-CD276 antibody [EPNCIR122] (ab218280)
    • Alexa Fluor® 594 Anti-CD276 antibody [EPNCIR122] (ab218282)
    • Alexa Fluor® 555 Anti-CD276 antibody [EPNCIR122] (ab218283)
    • Anti-CD276 antibody [EPNCIR122] - BSA and Azide free (ab256585)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081)
    • Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human CD276 knockout HEK-293T cell line (ab266658)
  • Recombinant Protein

    • Recombinant human CD276 protein (Fc Chimera Active) (ab216204)
  • Related Products

    • Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab134161 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-Fr
Use at an assay dependent concentration.
WB
1/200. Predicted molecular weight: 57 kDa.
IP
1/10 - 1/100.
Flow Cyt
1/1000 - 1/10000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF
Use a concentration of 1 µg/ml.
Notes
IHC-Fr
Use at an assay dependent concentration.
WB
1/200. Predicted molecular weight: 57 kDa.
IP
1/10 - 1/100.
Flow Cyt
1/1000 - 1/10000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF
Use a concentration of 1 µg/ml.
Application notes
Is unsuitable for IHC-P.

Target

  • Function

    May participate in the regulation of T-cell-mediated immune response. May play a protective role in tumor cells by inhibiting natural-killer mediated cell lysis as well as a role of marker for detection of neuroblastoma cells. May be involved in the development of acute and chronic transplant rejection and in the regulation of lymphocytic activity at mucosal surfaces. Could also play a key role in providing the placenta and fetus with a suitable immunological environment throughout pregnancy. Both isoform 1 and isoform 2 appear to be redundant in their ability to modulate CD4 T-cell responses. Isoform 2 is shown to enhance the induction of cytotoxic T-cells and selectively stimulates interferon gamma production in the presence of T-cell receptor signaling.
  • Tissue specificity

    Ubiquitous but not detectable in peripheral blood lymphocytes or granulocytes. Weakly expressed in resting monocytes. Expressed in dendritic cells derived from monocytes. Expressed in epithelial cells of sinonasal tissue. Expressed in extravillous trophoblast cells and Hofbauer cells of the first trimester placenta and term placenta.
  • Sequence similarities

    Belongs to the immunoglobulin superfamily. BTN/MOG family.
    Contains 2 Ig-like C2-type (immunoglobulin-like) domains.
    Contains 2 Ig-like V-type (immunoglobulin-like) domains.
  • Cellular localization

    Membrane.
  • Target information above from: UniProt accession Q5ZPR3 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 80381 Human
    • Entrez Gene: 102657 Mouse
    • Omim: 605715 Human
    • SwissProt: Q5ZPR3 Human
    • SwissProt: Q8VE98 Mouse
    • Unigene: 77873 Human
    • Unigene: 5356 Mouse
    • Alternative names

      • 4Ig B7 H3 antibody
      • 4Ig-B7-H3 antibody
      • AU016588 antibody
      • B7 H3 antibody
      • B7 homolog 3 antibody
      • B7-H3 antibody
      • B7H3 antibody
      • B7RP-2 antibody
      • CD_antigen=CD276 antibody
      • CD276 antibody
      • CD276 antigen antibody
      • CD276 molecule antibody
      • CD276_HUMAN antibody
      • Costimulatory molecule antibody
      • Flags: Precursor antibody
      • PSEC0249 antibody
      • UNQ309/PRO352 antibody
      see all

    Images

    • Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
      All lanes : Anti-CD276 antibody [EPNCIR122] (ab134161) at 1/1000 dilution

      Lane 1 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate boiled. with NFDM/TBST
      Lane 2 : HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate unboiled. with NFDM/TBST
      Lane 3 : LNCaP (Human prostate carcinoma epithelial cell) whole cell lysate boiled. with NFDM/TBST
      Lane 4 : LNCaP (Human prostate carcinoma epithelial cell) whole cell lysate unboiled. with NFDM/TBST

      Lysates/proteins at 20 µg per lane.

      Blocking peptides at 5 % per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution (Goats anti-rabbit IgG (H+L) Peroxidase Conjugated)

      Predicted band size: 57 kDa
      Observed band size: 90 kDa why is the actual band size different from the predicted?



      We recommend not to boil the samples after lysis to get desired WB results.

      CD276 (B7-H3) has a molecular weight of about 90–100 kDa, depending on the glycosylation levels. [PMID:22473715].

    • Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Anti-CD276 antibody [EPNCIR122] (ab134161) at 1/200 dilution + CHO (hamster ovary epithelial cell) whole cell lysates transfected with human CD276 at 15 µg

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

      Predicted band size: 57 kDa
      Observed band size: 110 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking/dilution buffer: 5% NFDM/TBST

      Performed using purified antibody.

    • Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (ab134161)

      Flow cytometry overlay histogram showing wild-type HEK293 (green line) and CD276 knockout HEK293 (ab266658) cells stained with ab134161 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab134161) (1x106 in 100μl at 0.2 μg/ml) for 30 min at 4°C.

      The secondary antibody Goat anti-rabbit IgG H&L (Alexa Fluor® 488, pre-adsorbed) (ab150081) was used at 1/2000 for 30 min at 4°C.

      Isotype control antibody was Rabbit IgG (monoclonal) (ab172730) used at the same concentration and conditions as the primary antibody (wild-type HEK293 - black line CD276 HEK293 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

      Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

    • Immunocytochemistry/ Immunofluorescence - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Immunocytochemistry/ Immunofluorescence - Anti-CD276 antibody [EPNCIR122] (ab134161)
      ab134161 staining CD276 in wild-type HEK293 cells (top panel) and CD276 knockout HEK293 cells (ab266658) (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab134161 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
      Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).
    • Immunoprecipitation - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Immunoprecipitation - Anti-CD276 antibody [EPNCIR122] (ab134161)

      CD276 was immunoprecipitated from 10 ug of HEK 293 (human embryonic kidney epithelial cell) whole cell lysate with ab134161 at 1/40 diltuion. Western blot was permformed from the immunoprecipitate using ab 134161 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/500 dilution.

      Lane 1: HEK 293 whole cell lysate 10 ug (Input).

      Lane 2: ab134161 IP in HEK 293 whole cell lysate.

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab134161 in HEK 293 whole cell lysate.

      Blocking/dilution buffer: 5% NFDM/TBST

      Performed using purified antibody.

    • Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Anti-CD276 antibody [EPNCIR122] (ab134161) at 1/1000 dilution + LNCap (human prostate carcinoma epithelial cell) whole cell lysate at 15 µg

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

      Predicted band size: 57 kDa
      Observed band size: 110 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking/dilution buffer: 5% NFDM/TBST

      Performed using purified antibody.

    • Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (ab134161)

      Overlay histogram showing THP-1 (human monocytic leukemia monocyte) cells stained with ab134161 (red line). The cells were fixed with 4% paraformaldehyde and then permeabilized with 90% methanol. The cells were incubated with the antibody (ab134161) at 1/80 dilution. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution. Isotype control antibody (black line) was rabbit monoclonal IgG (ab172730) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

      Performed using purified antibody.

    • Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Flow Cytometry - Anti-CD276 antibody [EPNCIR122] (ab134161)

      Overlay histogram showing THP1 cells stained with ab134161 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% human serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab134161, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

    • Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Western blot - Anti-CD276 antibody [EPNCIR122] (ab134161)
      All lanes : Anti-CD276 antibody [EPNCIR122] (ab134161) at 0.264 µg/ml

      Lane 1 : CHO-K1 cell lysates, transfected with Human CD276
      Lane 2 : CHO-K1 cell lysates, transfected with Mouse CD276.

      Lysates/proteins at 20 µg per lane.

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

      Predicted band size: 57 kDa


      Exposure time: 3 minutes


      Blocking/dilution buffer: 5% NFDM/TBST

    • Immunohistochemistry (Frozen sections) - Anti-CD276 antibody [EPNCIR122] (ab134161)
      Immunohistochemistry (Frozen sections) - Anti-CD276 antibody [EPNCIR122] (ab134161)Image from Seaman S et al. Cancer Cell, 31, 501-515. Fig2.D doi:10.1016/j.ccell.2017.03.005. with permission from Elsevier

      Immunohistochemistry of mouse MC38 colon liver metastasis. Staining CD276 with ab134161 (green). Normal tissue (N)/tumor tissue (T) margins are indicated by a white dash.

       

    • Anti-CD276 antibody [EPNCIR122] (ab134161)
      Anti-CD276 antibody [EPNCIR122] (ab134161)

    Protocols

    • Flow cytometry protocols
    • Immunoprecipitation protocols
    • Immunohistochemistry protocols
    • Western blot protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (12)

    Publishing research using ab134161? Please let us know so that we can cite the reference in this datasheet.

    ab134161 has been referenced in 12 publications.

    • Hu X  et al. B7-H3, Negatively Regulated by miR-128, Promotes Colorectal Cancer Cell Proliferation and Migration. Cell Biochem Biophys 79:397-405 (2021). PubMed: 33743142
    • Bachawal S  et al. Evaluation of ductal carcinoma in situ grade via triple-modal molecular imaging of B7-H3 expression. NPJ Breast Cancer 6:14 (2020). PubMed: 32377564
    • Zhang H  et al. Survival Association and Cell Cycle Effects of B7H3 in Neuroblastoma. J Korean Neurosurg Soc 63:707-716 (2020). PubMed: 32580265
    • Du H  et al. Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells. Cancer Cell 35:221-237.e8 (2019). PubMed: 30753824
    • Xu ZL  et al. B7-H3 promotes malignant progression of muscle-invasive bladder cancer. Oncol Rep 40:2722-2733 (2018). PubMed: 30132557
    View all Publications for this product

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