Overview

  • Product name

    Rat TNF alpha ELISA Kit
    See all TNF alpha kits
  • Detection method

    Colorimetric
  • Precision

    Intra-assay
    Sample n Mean SD CV%
    Supernatant 3 3.9%
    Inter-assay
    Sample n Mean SD CV%
    Supernatant 8 5.1%
  • Sample type

    Cell culture supernatant, Serum, Heparin Plasma, EDTA Plasma, Citrate Plasma
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    1.04 pg/ml
  • Range

    18.75 pg/ml - 1200 pg/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 106 101% - 115%
    Serum 81 79% - 83%
    Heparin Plasma 77 76% - 78%
    EDTA Plasma 81 81% - 82%
    Citrate Plasma 82 80% - 83%

  • Assay time

    1h 30m
  • Assay duration

    One step assay
  • Species reactivity

    Reacts with: Rat
  • Product overview

    TNF alpha in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of TNF alpha protein in rat serum, plasma, and cell culture supernatants.


    The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB Development Solution is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.


    Tumor necrosis factor, or TNF alpha, is a cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. TNF alpha is a ligand of the TNF superfamily which plays a central role in inflammation, apoptosis, proliferation, invasion, angiogenesis, metastasis, and morphogenesis. It is mainly secreted by macrophages, and can induce cell death of certain tumor cell lines. Mouse and Human TNF alpha are 95% and 79% identical to Rat TNF alpha, respectively.


    TNF alpha is expressed on macrophages and endothelial, epithelial, and tumor cells as a 26 kDa transmembrane protein. TNF-alpha is cleaved by proteolytic processing into six chains: (1) TNF membrane form, (2) Intracellular domain 1 (ICD1), (3) Intracellular domain 2 (ICD2), (4) C-domain 1, (5) C-domain 2 and (6) TNF soluble form. ICD1 and ICD2 are released into the cytosol, while C-domain 1 and C-domain 2 are released into the extracellular space. Only the membrane form is phosphorylated on serine residues. Dephosphorylation of the membrane form occurs by binding to soluble TNFRSF1A/TNFR1.


    Signaling from TNF-alpha differs depending on the type of ligand initiating the signaling event (intracellular, membrane, or soluble). This is demonstrated by the membrane form of TNF-alpha mediates anti-tumorigenic therapeutic responses whereas the soluble ligand is linked to inflammation and proliferation. Likewise, the TNF intracellular domain (ICD) form induces IL-12 production in dendritic cells.


     

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Pre-coated microplate (12 x 8 well strips)

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 1 x 96 tests
    10X Wash Buffer PT (ab206977) 1 x 20ml
    Antibody Diluent 4BR 1 x 6ml
    Plate Seals 1 unit
    10X Rat TNF alpha Capture Antibody 1 x 600µl
    10X Rat TNF alpha Detector Antibody 1 x 600µl
    Rat TNF alpha Lyophilized Recombinant Protein 2 vials
    Sample Diluent NS (ab193972) 1 x 50ml
    SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit
    Stop Solution 1 x 12ml
    TMB Development Solution 1 x 12ml
  • Research areas

  • 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
  • Alternative names

    • APC1
    • APC1 protein
    • Cachectin
    • DIF
    • Differentiation inducing factor
    • Macrophage cytotoxic factor
    • Tnf
    • TNF superfamily member 2
    • TNF superfamily, member 2
    • TNF, macrophage derived
    • TNF, monocyte derived
    • TNF-a
    • TNF-alpha
    • TNFA
    • TNFA_HUMAN
    • TNFSF2
    • Tumor necrosis factor
    • Tumor necrosis factor (TNF superfamily member 2)
    • Tumor necrosis factor alpha
    • Tumor necrosis factor ligand superfamily member 2
    • Tumor Necrosis Factor, Membrane Form
    • Tumor necrosis factor, soluble form
    see all
  • Database links

Associated products

Applications

Our Abpromise guarantee covers the use of ab236712 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
Sandwich ELISA Use at an assay dependent concentration.

Images

  • SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.

     

  • The TNF alpha standard curve was prepared as described in Section 10. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed.

  • The concentrations of TNF alpha were measured in duplicates, interpolated from the TNF alpha standard curves and corrected for sample dilution. Undiluted samples are 50 ng/mL PMA + 500 ng/mL calcium ionomycin treated splenocyte cell culture supernatant - 100%. Treatment length was 72 hours. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean TNF alpha concentration was determined to be 156.05 pg/mL in treated splenocyte culture supernatant.

  • The concentrations of TNF alpha were measured in duplicate and interpolated from the TNF alpha standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean TNF alpha concentration was below the lowest standard (<18.75) pg/mL in unstimulated splenocyte cell culture supernatant and 156.05 pg/mL in stimulated splenocyte cell culture supernatant. Stimulated samples were treated with 50 ng/mL PMA + 500 ng/mL calcium ionomycin for 72 hours. Unstimulated cells as a control were cultured in parallel for 72 hours.

Protocols

References

ab236712 has not yet been referenced specifically in any publications.

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