RecombinantRabMAb

Recombinant Anti-TNF alpha antibody [EPR20972] (ab215188)

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Western blot - Anti-TNF alpha antibody [EPR20972] (ab215188)
  • Immunocytochemistry/ Immunofluorescence - Anti-TNF alpha antibody [EPR20972] (ab215188)
  • Flow Cytometry - Anti-TNF alpha antibody [EPR20972] (ab215188)
  • Western blot - Anti-TNF alpha antibody [EPR20972] (ab215188)
  • Immunoprecipitation - Anti-TNF alpha antibody [EPR20972] (ab215188)
  • Anti-TNF alpha antibody [EPR20972] (ab215188)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR20972] to TNF alpha
  • Suitable for: WB, ICC/IF, Flow Cyt, IP
  • Reacts with: Mouse, Human

Overview

  • Product name

    Anti-TNF alpha antibody [EPR20972]
    See all TNF alpha primary antibodies

  • Description

    Rabbit monoclonal [EPR20972] to TNF alpha

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, ICC/IF, Flow Cyt, IPmore details

  • Species reactivity

    Reacts with: Mouse, Human

  • Immunogen

    Recombinant fragment within Mouse TNF alpha aa 1 to the C-terminus. The exact sequence is proprietary.
    Database link: P06804

  • Positive control

    • WB: RAW 264.7 treated with lipopolysaccharides (LPS) with addition of brefeldin A (BFA) whole cell lysate; THP-1 differentiated with TPA overnight, then treated with LPS for 7 hours with addition of BFA for the last 3 hours, whole cell lysate. ICC/IF: RAW 264.7 cells treated with LPS with addition of BFA. Flow Cyt: Mouse splenocytes treated with 20ng/ml PMA, 1µg/ml Ionomycin and 10µM BFA. IP: RAW 264.7 treated with LPS with addition of BFA whole cell lysate

  • 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.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Properties

Applications

Our Abpromise guarantee covers the use of ab215188 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 33, 26, 17 kDa (predicted molecular weight: 26 kDa).
ICC/IF 1/100.
Flow Cyt 1/600.

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

 
IP 1/30.

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 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 (TNF superfamily member 2) 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 [EPR20972] (ab215188)
      Western blot - Anti-TNF alpha antibody [EPR20972] (ab215188)
      All lanes : Anti-TNF alpha antibody [EPR20972] (ab215188) at 1/1000 dilution

      Lane 1 : Untreated RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus), whole cell lysate
      Lane 2 : Untreated RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 µg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate

      Lysates/proteins at 10 µg per lane.

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

      Developed using the ECL technique.

      Predicted band size: 26 kDa
      Observed band size: 17,26,33 kDa
      why is the actual band size different from the predicted?


      Exposure time: 15 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Immunocytochemistry/ Immunofluorescence - Anti-TNF alpha antibody [EPR20972] (ab215188)
      Immunocytochemistry/ Immunofluorescence - Anti-TNF alpha antibody [EPR20972] (ab215188)

      Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cells, untreated or treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours labeling TNF alpha with ab215188 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Cytoplasmic staining was increased on RAW 264.7 cells when treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours.

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

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

    • Flow Cytometry - Anti-TNF alpha antibody [EPR20972] (ab215188)
      Flow Cytometry - Anti-TNF alpha antibody [EPR20972] (ab215188)

      Flow cytometric analysis of 4% paraformaldehyde-fixed, 0.1% Tween-20 permeabilized mouse splenocytes treated with 20 ng/ml PMA, 1 μg/ml Ionomycin and 10 μM Brefeldin A for 6 hours labeling TNF alpha with ab215188 at 1/600 dilution (right panel) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left panel). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/2000 dilution was used as the secondary antibody.

      Cells were surface stained with anti-mouse CD3, fixed with 4% PFA for 10 minutes, then permeabilized with 0.1% Tween-20 and intracellular stained with anti-rabbit IgG and ab215188. TNF alpha is mainly expressed in T cells (CD3+ population) while only a small population of CD3- cells can express TNF-alpha.

    • Western blot - Anti-TNF alpha antibody [EPR20972] (ab215188)
      Western blot - Anti-TNF alpha antibody [EPR20972] (ab215188)
      All lanes : Anti-TNF alpha antibody [EPR20972] (ab215188) at 1/1000 dilution

      Lane 1 : THP-1 (human monocytic leukemia cell line) differentiated with 100 nM TPA overnight, whole cell lysate
      Lane 2 : THP-1 (human monocytic leukemia cell line) differentiated with 100 nM TPA overnight, then treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 µg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

      Developed using the ECL technique.

      Predicted band size: 26 kDa
      Observed band size: 26 kDa


      Exposure time: 3 minutes


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Immunoprecipitation - Anti-TNF alpha antibody [EPR20972] (ab215188)
      Immunoprecipitation - Anti-TNF alpha antibody [EPR20972] (ab215188)

      TNF alpha was immunoprecipitated from 0.35 mg of RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate with ab215188 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab215188 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

      Lane 1: RAW 264.7 treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate 10 µg (Input). 

      Lane 2: ab215188 IP in RAW 264.7 treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate (+). 

      Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab215188 in RAW 264.7 treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate (-).

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

      Exposure time: 3 seconds.

    • Anti-TNF alpha antibody [EPR20972] (ab215188)
      Anti-TNF alpha antibody [EPR20972] (ab215188)

    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 (2)

    Publishing research using ab215188? Please let us know so that we can cite the reference in this datasheet.

    ab215188 has been referenced in 2 publications.

    • Mai W  et al. Berberine Inhibits Nod-Like Receptor Family Pyrin Domain Containing 3 Inflammasome Activation and Pyroptosis in Nonalcoholic Steatohepatitis via the ROS/TXNIP Axis. Front Pharmacol 11:185 (2020). PubMed: 32194416
    • Wang Z  et al. TRPV4-induced inflammatory response is involved in neuronal death in pilocarpine model of temporal lobe epilepsy in mice. Cell Death Dis 10:386 (2019). PubMed: 31097691

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