Anti-TNF alpha antibody (ab9635)

Reviews (3)Q&A (5)References (24)

Overview

  • Product name
    Anti-TNF alpha antibody
    See all TNF alpha primary antibodies
  • Description
    Rabbit polyclonal to TNF alpha
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, WB, ELISA, Neutralising, Sandwich ELISAmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant fragment corresponding to Human TNF alpha aa 77-233. E.coli derived Recombinant Human TNF-alpha
    Database link: P01375

  • Positive control
    • WB: Recombinant human TNF alpha protein (ab9642), SW480 cell lysate. IHC-P: Human tonsil tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab9635 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 Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
WB Use at an assay dependent concentration. To detect TNF-alpha by Western Blot analysis this antibody can be used at a concentration of 0.1 - 0.2 µg/ml. Used in conjunction with compatible secondary reagents the detection limit for recombinant TNF-alpha is 1.5 - 3.0 ng/lane, under either reducing or non-reducing conditions.
ELISA Use at an assay dependent concentration. Can be paired for ELISA with Mouse monoclonal to TNF alpha (ab9348). To detect TNF-alpha by direct ELISA (using 100µl/well antibody solution) a concentration of at least 0.5µg/ml of this antibody is required. This antigen affinity purified antibody, in conjunction with compatible secondary reagents, allows the detection of 0.2 - 0.4 ng/well of recombinant TNF-alpha.
Neutralising Use at an assay dependent concentration. To yield one-half maximal inhibition [ND50] of the biological activity of hTNF-alpha (0.5 ng/ml), a concentration of 0.08 - 0.10 µg/ml of this antibody is required.
Sandwich ELISA Use a concentration of 0.5 µg/ml. Can be paired for Sandwich ELISA with Mouse monoclonal [2C8] to TNF alpha (ab8348).

For sandwich ELISA, use this antibody as Detection at 0.5µg/ml with ab8348 as Capture.

Target

  • Function
    Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation.
  • Involvement in disease
    Genetic variations in TNF are a cause of susceptibility psoriatic arthritis (PSORAS) [MIM:607507]. PSORAS is an inflammatory, seronegative arthritis associated with psoriasis. It is a heterogeneous disorder ranging from a mild, non-destructive disease to a severe, progressive, erosive arthropathy. Five types of psoriatic arthritis have been defined: asymmetrical oligoarthritis characterized by primary involvement of the small joints of the fingers or toes; asymmetrical arthritis which involves the joints of the extremities; symmetrical polyarthritis characterized by a rheumatoidlike pattern that can involve hands, wrists, ankles, and feet; arthritis mutilans, which is a rare but deforming and destructive condition; arthritis of the sacroiliac joints and spine (psoriatic spondylitis).
  • Sequence similarities
    Belongs to the tumor necrosis factor family.
  • Post-translational
    modifications
    The soluble form derives from the membrane form by proteolytic processing.
    The membrane form, but not the soluble form, is phosphorylated on serine residues. Dephosphorylation of the membrane form occurs by binding to soluble TNFRSF1A/TNFR1.
    O-glycosylated; glycans contain galactose, N-acetylgalactosamine and N-acetylneuraminic acid.
  • Cellular localization
    Secreted and Cell membrane.
  • Information by UniProt
  • Database links
    • Alternative names
      • APC1 antibody
      • APC1 protein antibody
      • Cachectin antibody
      • DIF antibody
      • Differentiation inducing factor antibody
      • Macrophage cytotoxic factor antibody
      • Tnf antibody
      • TNF superfamily, member 2 antibody
      • TNF, macrophage derived antibody
      • TNF, monocyte derived antibody
      • TNF-a antibody
      • TNF-alpha antibody
      • TNFA antibody
      • TNFA_HUMAN antibody
      • TNFSF2 antibody
      • Tumor necrosis factor alpha antibody
      • Tumor necrosis factor antibody
      • Tumor necrosis factor ligand superfamily member 2 antibody
      • Tumor Necrosis Factor, Membrane Form antibody
      • Tumor necrosis factor, soluble form antibody
      see all

    Images

    • Western blot - Anti-TNF alpha antibody (ab9635)
      Western blot - Anti-TNF alpha antibody (ab9635)
      Anti-TNF alpha antibody (ab9635) at 0.2 µg/ml + SW480 (Human colon adenocarcinoma cell line) Whole Cell Lysate at 10 µg

      Secondary
      Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

      Observed band size: 26 kDa
      why is the actual band size different from the predicted?
      Additional bands at: 48 kDa. We are unsure as to the identity of these extra bands.

    • Sandwich ELISA - Anti-TNF alpha antibody (ab9635)
      Sandwich ELISA - Anti-TNF alpha antibody (ab9635)
      Standard curve for TNF alpha (Analyte: ab9642); dilution range 1pg/ml to 1µg/ml using Capture Antibody Mouse monoclonal [2C8] to TNF alpha (ab8348) at 5µg/ml and Detector Antibody Rabbit polyclonal to TNF alpha (ab9635) at 0.5µg/ml.
    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody (ab9635)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody (ab9635)

      IHC image of TNF alpha staining in human tonsil formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab9635, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

      For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

    • Western blot - Anti-TNF alpha antibody (ab9635)
      Western blot - Anti-TNF alpha antibody (ab9635)
    • Western blot
      Western blot

    References

    This product has been referenced in:
    • Zheng Y  et al. MicroRNA-24 inhibits the proliferation and migration of endothelial cells in patients with atherosclerosis by targeting importin-a3 and regulating inflammatory responses. Exp Ther Med 15:338-344 (2018). Read more (PubMed: 29250154) »
    • Maleki SN  et al. Berberine confers neuroprotection in coping with focal cerebral ischemia by targeting inflammatory cytokines. J Chem Neuroanat 87:54-59 (2018). Read more (PubMed: 28495517) »
    See all 24 Publications for this product

    Publishing research using ab9635? Please let us know so that we can cite the reference in this datasheet.

    Customer reviews and Q&As

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    1-8 of 8 Abreviews or Q&A

    Question

    Kindly contacted us for informaton about use of these products in ELISA.

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    Answer

    In order to detect Human TNF-α by sandwich ELISA (using 100μl/well antibody solution) a concentration of 0.5 - 2.0 μg/ml of this antibody is required. We recommend determining optimal dilutions of this antibody for your ELISA assay.
    The following paper may of use to you for the ELISA protocol used with this antibody:

    Our references for Anti-Human TNF-α (500-P31A) include one article citing use in ELISA:
    Halin, C, Synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha. Cancer Research; 63(12):3202-3210. 15 Jun 2003 (PubMed ID: 12810649).

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    Question

    Inquiry: Hello We would like to develop an Electrochemical Immunosensor using a sandwich type assay (Primary Antibody-Antigen- Secondary antibody) and we need to choose a suitable pair of Ab with high affinity reaction with their Antigen: TNF alpha protein for Immunoassay applications.. We would like to know the code of suitable primary and secondary antibodies. that you recommend us Thank you very much. Regards

    Read More
    Answer

    Thank you for contacting us.

    We have in our catalogue the following pairs of antibodies that have been tested for the detection of Human TNF-alpha in sELISA :

    Anti-TNF alpha antibody [2C8] (https://www.abcam.com/ab8348) as capture + Rabbit polyclonal to TNF alpha (https://www.abcam.com/ab9635) as detection. Please note that these 2 antibodies are unconjugated.
    Anti-TNF alpha antibody [2C8] (https://www.abcam.com/ab8348) as capture + Anti-TNF alpha antibody [F6C5 ] (HRP) (https://www.abcam.com/ab24473) as detection.



    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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    Question

    On your website you give AB9635 as a good candidate for sandwich assay with AB8348. I would like to use that combination in my PhD. The goal is to detect TNF-alpha in cell culture medium with high specificity and even when the antigen is present in low concentrations. That’s why information about cross reactivity is important. Do you have any information about proteins or other molecules that can cross react with the antibody pair?

    I was also wondering if there is a reason that the ELISA-kits don’t use the antibodies you sell individually. It would be a nice comparison to make use of an ELISA kit with the AB8348 and AB9635 antibodies.

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    Answer

    Thank you for your inquiry.

    We haven't specifically tested ab8348 and ab9635 for cross-reactivity with other proteins.

    For ab9635, I BLASTed the immunogen sequence against other human proteins and no significant homology was detected (< 34 %) so we wouldn't anticipate cross-reactivity. Plus this is produced from sera of rabbits pre-immunized with highly pure recombinant Human TNF-α and it was purified by affinity chromatography employing immobilized Human TNF-α matrix.

    We have some references for both antibodies that you may be interested in:

    https://www.abcam.com/TNF-alpha-antibody-ab9635-references.html

    https://www.abcam.com/TNF-alpha-antibody-2C8-ab8348-references.html

    The protocol for using the antibodies together in sELISA can be found here: https://www.abcam.com/ps/products/8/ab8348/documents/Protocol%20rabbit.pdf

    The ELISA kits we sell may come from different sources, and sometimes we are not provided with the individual antibodies for sale. We can always check individual kits to see if we have the antibodies, but in most cases they probably aren't available.

    I hope this information helps. Please contact us with any other questions.

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    Question

    searching for antibody blocking TNF alpha on human cultured cells - blocking free and membrane bound?

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    Answer

    Merci pour votre patience. Veuillez trouver en attaché le document que le laboratoire m'avait envoyé pour le ab9635. Il montre que l'anticorps est efficace à inhiber le TNF alpha à moins que 0.1ug/ml. Veuillez ne pas hésiter à nous recontacter si vous avez besoin de plus d'informations. Je vous souhaite bon succès dans votre recherche.

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    ELISA abreview for Anti-TNF alpha antibody

     Excellent
    Abreviews
    Abcam guarantees this product to work in the species/application used in this Abreview.
    abreview image
    Application
    ELISA
    Sample
    Human Recombinant protein (expressed in E.coli strain BL21(DE3))
    Specification
    expressed in E.coli strain BL21(DE3)
    Blocking step
    Milk as blocking agent for 20 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
    Type
    Indirect
    Read More

    Abcam user community

    Verified customer

    Submitted Oct 12 2010

    Western blot abreview for Anti-TNF alpha antibody

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Escherichia coli Purified protein (BL21(DE3))
    Loading amount
    1 µg
    Specification
    BL21(DE3)
    Gel Running Conditions
    Reduced Denaturing (4-20%)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More

    Abcam user community

    Verified customer

    Submitted Aug 26 2010

    Western blot abreview for Anti-TNF alpha antibody

     Excellent
    Abreviews
    Abcam guarantees this product to work in the species/application used in this Abreview.
    abreview image
    Application
    Western blot
    Sample
    Human Recombinant protein (Recombinant Human TFN-alpha (Source: E coil))
    Loading amount
    0.5 µg
    Specification
    Recombinant Human TFN-alpha (Source: E coil)
    Gel Running Conditions
    Reduced Denaturing (4-20%)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C
    Read More

    Abcam user community

    Verified customer

    Submitted Feb 15 2010

    Question

    Can you tell me how the neutralization experiment was done?

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    Answer

    Thank you for your enquiry and your patience. The neutralization experiment was conducted as follows: Procedure: 1) Resuspend 929 cells in regular culture media. Transfer 100ml/well in assay plate (8,100 cells/well). Incubate 37oC overnight. 2) In separate plate, serial dilute a-h-TNF-a in assay media containing 2mg/ml Actinomycin D and 10% horse serum in triplicate. Add 60ml/well assay media containing 2ng/ml h-TNF-a resulting in 1:2 dilution. Incubate 1 hour. 3) Transfer 100ml/well h-TNF-a/anti-h-TNF-a complex to cells in assay plate Final volume/well is 200ml containing 1mg/ml Actinomycin D, 0.5 ng/ml h-TNF-a and anti-h-TNF-a as indicated below. 4) Incubate 20 hours. 5) Add 20ml of Promega Substrate CellTiter 96 Aqueous One Solution Reagent to each well. 6) Incubate 37oC and read OD at 490 nm. Data: Conc. anti-h-TNF-a O.D. reading at 490nm after 4 hours ug/ml row 1 row 2 row 3 Ave. O.D. Net O.D. 25.00 12.50 6.25 3.13 1.56 0.78 0.39 0.20 0.10 0.00 I hope this information helps, please do not hesitate to contact us if you need any more advice or information,

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