Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-CD40 antibody [EPR20735] (ab224639)

Submit a review Submit a question References (4)

Overview

  • Product name

    Anti-CD40 antibody [EPR20735]
    See all CD40 primary antibodies

  • Description

    Rabbit monoclonal [EPR20735] to CD40

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IHC-P, Flow Cyt, IP, ICC/IFmore details

  • Species reactivity

    Reacts with: Human

  • Immunogen

    Recombinant fragment within Human CD40 aa 1-200. The exact sequence is proprietary.
    Database link: P25942

  • Positive control

    • WB: Human lymph node and lymphoma tissue lysates; Raji whole cell lysate. IHC-P: Human tonsil, Hodgkin's lymphoma and diffuse large B-cell lymphoma tissues. ICC/IF: Raji cells. Flow Cyt: Raji cells. IP: Raji whole cell lysate. ICC/IF KO: U2OS cells (U2OS-CD40 knockout cells used as negative cell line)

  • 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 ab224639 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 42 kDa (predicted molecular weight: 30 kDa).
IHC-P 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Flow Cyt 1/600.

ab172730-Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP 1/30.
ICC/IF 1/100.

Target

  • Function

    Receptor for TNFSF5/CD40LG.

  • Tissue specificity

    B-cells and in primary carcinomas.

  • Involvement in disease

    Defects in CD40 are the cause of hyper-IgM immunodeficiency syndrome type 3 (HIGM3) [MIM:606843]; also known as hyper-IgM syndrome 3. HIGM3 is an autosomal recessive disorder which includes an inability of B cells to undergo isotype switching, one of the final differentiation steps in the humoral immune system, an inability to mount an antibody-specific immune response, and a lack of germinal center formation.

  • Sequence similarities

    Contains 4 TNFR-Cys repeats.

  • Cellular localization

    Secreted and Cell membrane.
  • Information by UniProt

  • Database links

    • Alternative names

      • AI326936 antibody
      • B cell associated molecule CD40 antibody
      • B cell surface antigen CD40 antibody
      • B cell-associated molecule antibody
      • B-cell surface antigen CD40 antibody
      • Bp50 antibody
      • CD 40 antibody
      • CD40 antibody
      • CD40 antigen (TNF receptor superfamily member 5) antibody
      • CD40 antigen antibody
      • CD40 molecule antibody
      • CD40 molecule, TNF receptor superfamily member 5 antibody
      • CD40 protein antibody
      • CD40 type II isoform antibody
      • CD40L receptor antibody
      • CDw40 antibody
      • GP39 antibody
      • HIGM1 antibody
      • IGM antibody
      • IMD3 antibody
      • MGC9013 antibody
      • Nerve growth factor receptor related B lymphocyte activation molecule antibody
      • OTTHUMP00000031699 antibody
      • OTTHUMP00000031700 antibody
      • p50 antibody
      • T-BAM antibody
      • TBAM antibody
      • TNF receptor superfamily member 5 antibody
      • TNFRSF5 antibody
      • TNR5_HUMAN antibody
      • TRAP antibody
      • Tumor necrosis factor receptor superfamily , member 5 antibody
      • Tumor necrosis factor receptor superfamily member 5 antibody
      • Tumor necrosis factor receptor superfamily member 5 precursor antibody
      • Tumor necrosis factor receptor superfamily, member 5, isoform CRA_a antibody
      see all

    Images

    • Immunocytochemistry/ Immunofluorescence - Anti-CD40 antibody [EPR20735] (ab224639)
      Immunocytochemistry/ Immunofluorescence - Anti-CD40 antibody [EPR20735] (ab224639)

      ab224639 staining CD40 in wild-type U2OS cells (top panel) and CD40 knockout U2OS cells (bottom panel). The cells were fixed with PFA (10min), 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 ab224639 at 1/100 dilution and ab7291 (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 pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

      Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

    • Flow Cytometry - Anti-CD40 antibody [EPR20735] (ab224639)
      Flow Cytometry - Anti-CD40 antibody [EPR20735] (ab224639)

      Flow cytometric analysis of Raji (human Burkitt's lymphoma cell line) cell line labeling CD40 with ab224639 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077), at 1/2000 dilution was used as the secondary antibody.

      Gated on total viable cells.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20735] (ab224639)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20735] (ab224639)

      Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD40 with ab224639 at 1/250 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous and cytoplasmic staining in human tonsil tissue (PMID:20593977; PMID:7507299) is observed. Counter stained with hematoxylin.

      Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    • Immunocytochemistry/ Immunofluorescence - Anti-CD40 antibody [EPR20735] (ab224639)
      Immunocytochemistry/ Immunofluorescence - Anti-CD40 antibody [EPR20735] (ab224639)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Raji (human Burkitt's lymphoma cell line) cells labeling CD40 with ab224639 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous and weakly cytoplasmic staining in Raji cell line.

      Negative control: Jurkat T cell line (PMID:10498643).

      The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

      Secondary antibody only control: PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

    • Western blot - Anti-CD40 antibody [EPR20735] (ab224639)
      Western blot - Anti-CD40 antibody [EPR20735] (ab224639)
      All lanes : Anti-CD40 antibody [EPR20735] (ab224639) at 1/1000 dilution

      Lane 1 : Human lymph node tissue lysate
      Lane 2 : Human lymphoma tissue lysate
      Lane 3 : Raji (human Burkitt's lymphoma cell line) whole cell lysate

      Lysates/proteins at 20 µg per lane.

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

      Developed using the ECL technique.

      Predicted band size: 30 kDa
      Observed band size: 42 kDa
      why is the actual band size different from the predicted?


      Exposure time: 15 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Immunoprecipitation - Anti-CD40 antibody [EPR20735] (ab224639)
      Immunoprecipitation - Anti-CD40 antibody [EPR20735] (ab224639)

      CD40 was immunoprecipitated from 0.35 mg of Raji (human Burkitt's lymphoma cell line) whole cell lysate with ab224639 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab224639 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

      Lane 1: Raji whole cell lysate 10 μg (Input).
      Lane 2: ab224639 IP in Raji whole cell lysate (+).
      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab224639 in Raji whole cell lysate (-).

      Exposure time: 3 minutes.

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

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20735] (ab224639)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20735] (ab224639)

      Immunohistochemical analysis of paraffin-embedded human diffuse large B-cell lymphoma tissue labeling CD40 with ab224639 at 1/250 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining in human diffuse large B-cell lymphoma tissue (PMID: 20593977) is observed. Counter stained with hematoxylin.

      Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20735] (ab224639)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20735] (ab224639)

      Immunohistochemical analysis of paraffin-embedded human Hodgkin's lymphoma tissue labeling CD40 with ab224639 at 1/250 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous and cytoplasmic staining in Reed-Sternberg cells and tumor cells of human Hodgkin's lymphoma (PMID:7507299) is observed. Counter stained with hematoxylin.

      Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

      Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

    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 ab224639? Please let us know so that we can cite the reference in this datasheet.

    ab224639 has been referenced in 4 publications.

    • Zhou ZQ  et al. Adipose extracellular matrix promotes skin wound healing by inducing the differentiation of adipose-derived stem cells into fibroblasts. Int J Mol Med 43:890-900 (2019). PubMed: 30535488
    • Wang L  et al. Identification of circular RNA Hsa_circ_0001879 and Hsa_circ_0004104 as novel biomarkers for coronary artery disease. Atherosclerosis 286:88-96 (2019). PubMed: 31103880
    • Hong D  et al. Human-induced pluripotent stem cell-derived macrophages and their immunological function in response to tuberculosis infection. Stem Cell Res Ther 9:49 (2018). WB . PubMed: 29482598
    • Journo G  et al. Modulation of Cellular CpG DNA Methylation by Kaposi's Sarcoma-Associated Herpesvirus. J Virol 92:N/A (2018). PubMed: 29899086

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    There are currently no Customer reviews or Questions for ab224639.
    Please use the links above to contact us or submit feedback about this product.

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com