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    products/primary-antibodies/cd40-antibody-epr20540-ab213205.pdf

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Immunology Adaptive Immunity B Cells CD
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-CD40 antibody [EPR20540] (ab213205)

  • Datasheet
  • SDS
  • Certificate of Compliance
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Western blot - Anti-CD40 antibody [EPR20540] (ab213205)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)
  • Western blot - Anti-CD40 antibody [EPR20540] (ab213205)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)
  • Anti-CD40 antibody [EPR20540] (ab213205)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR20540] to CD40
  • Suitable for: IHC-P, WB
  • Knockout validated
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

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Recombinant human CD40 protein (Active) (ab155710)
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Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
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Anti-CD40 antibody [EPR20735] - BSA and Azide free (ab228818)

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Overview

  • Product name

    Anti-CD40 antibody [EPR20540]
    See all CD40 primary antibodies
  • Description

    Rabbit monoclonal [EPR20540] to CD40
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-P, WBmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Human tonsil, lymph node and lymphoma lysates; Raji, U-2 OS, and Daudi whole cell lysates. IHC-P: Human tonsil and large B cell lymphoma tissues.
  • 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

  • Form

    Liquid
  • Storage instructions

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

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR20540
  • Isotype

    IgG
  • Research areas

    • Immunology
    • Adaptive Immunity
    • B Cells
    • CD
    • Cardiovascular
    • Atherosclerosis
    • Vascular Inflammation
    • Innate and adaptive immunity
    • Stem Cells
    • Hematopoietic Progenitors
    • Lymphoid
    • B Lymphocytic Lineage
    • Stem Cells
    • Hematopoietic Progenitors
    • Myeloid
    • Dendritic Cell Lineage
    • Neuroscience
    • Processes

Associated products

  • Alternative Versions

    • Anti-CD40 antibody [EPR20540] - Low endotoxin, Azide free (ab223546)
    • Anti-CD40 antibody [EPR20540] - BSA and Azide free (ab271995)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Isotype control

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

    • Human CD40 knockout U-2 OS cell line (ab262486)
  • KO cell lysates

    • Human CD40 knockout U-2 OS cell lysate (ab263923)
  • Recombinant Protein

    • Recombinant human CD40 protein (Active) (ab155710)
  • Related Products

    • VeriBlot for IP Detection Reagent (HRP) (ab131366)
    • Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) (ab93684)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab213205 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
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB
1/2000. Detects a band of approximately 42 kDa (predicted molecular weight: 30 kDa).
Notes
IHC-P
1/1000. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB
1/2000. Detects a band of approximately 42 kDa (predicted molecular weight: 30 kDa).

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.
  • Target information above from: UniProt accession P25942 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 958 Human
    • Omim: 109535 Human
    • SwissProt: P25942 Human
    • Unigene: 472860 Human
    • 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

    • Western blot - Anti-CD40 antibody [EPR20540] (ab213205)
      Western blot - Anti-CD40 antibody [EPR20540] (ab213205)
      All lanes : Anti-CD40 antibody [EPR20540] (ab213205) at 1/2000 dilution

      Lane 1 : Wild-type U-2 OS whole cell lysate
      Lane 2 : CD40 knockout U-2 OS whole cell lysate
      Lane 3 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

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



      Lanes 1 - 3: Merged signal (red and green). Green - ab213205 observed at 42 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

      ab213205 was shown to react with CD40 in U-2 OS wild-type cells in Western blot. Loss of signal was observed when CD40 knockout sample was used. U-2 OS wild-type and CD40 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween®) before incubation with ab213205 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)

      Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling CD40 with ab213205 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. 

      Membranous and cytoplasmic staining on germinal center of human tonsil is observed [PMID: 10360965] [PMID: 7507299] [PMID:24452203].

      Counter stained with Hematoxylin.

      Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

      Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    • Western blot - Anti-CD40 antibody [EPR20540] (ab213205)
      Western blot - Anti-CD40 antibody [EPR20540] (ab213205)
      All lanes : Anti-CD40 antibody [EPR20540] (ab213205) at 1/2000 dilution

      Lane 1 : Human tonsil lysate
      Lane 2 : Human lymph node lysate
      Lane 3 : Human lymphoma lysate
      Lane 4 : Raji (Human Burkitt's lymphoma cell line) whole cell lysate
      Lane 5 : Daudi (Human Burkitt's lymphoma cell line) whole cell lysate
      Lane 6 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      Lanes 1-3 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/4000 dilution
      Lanes 4-6 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

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



      Blocking/Dilution buffer: 5% NFDM/TBST.

      Exposure time: Lane 1,2,3 and 4: 30 seconds; Lane 5,6 and 7: 5 seconds.

      The molecular weight observed is consistent with the literature (PMID: 23404288);

      Negative control: Jurkat (PMID:10498643; PMID:15708600).

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)

      Immunohistochemical analysis of paraffin-embedded human large B cell lymphoma tissue labeling CD40 with ab213205 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

      Membranous staining on human large B cell lymphoma is observed [PMID: 7507299].

      Counter stained with Hematoxylin.

      Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

      Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD40 antibody [EPR20540] (ab213205)

      Tissue Microarrays stained for "Anti-CD40 antibody [EPR20540]” using "ab213205" in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). The sections were incubated with ab213205 at +4°C overnight followed by Goat Anti-Rabbit IgG H&L (HRP) ab97051 at 1/500.

    • Anti-CD40 antibody [EPR20540] (ab213205)
      Anti-CD40 antibody [EPR20540] (ab213205)

    Protocols

    • Western blot protocols
    • Immunohistochemistry protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    Certificate of Compliance

    To download a Certificate of Compliance, please enter your Lot number below:

    References (0)

    Publishing research using ab213205? Please let us know so that we can cite the reference in this datasheet.

    ab213205 has not yet been referenced specifically in any publications.

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