Description

  • Product name

    Recombinant human TNF alpha protein
    See all TNF alpha proteins and peptides
  • Biological activity

    The ED50, as determined by the cytolysis of murine L929 cells in the presence of Actinomyocin D, is ≤ 0.05 ng/mL, corresponding to a specific activity of ≥ 2 x 107 units/mg.

  • Purity

    > 98 % SDS-PAGE.
    >98% by HPLC analyses. Sterile filtered.
  • Endotoxin level

    < 1.000 Eu/µg
  • Expression system

    Escherichia coli
  • Accession

  • Protein length

    Full length protein
  • Animal free

    No
  • Nature

    Recombinant
    • Species

      Human
    • Sequence

      VRSSSRTPSD KPVAHVVANP QAEGQLQWLN RRANALLANG VELRDNQLVV PSEGLYLIYS QVLFKGQGCP STHVLLTHTI SRIAVSYQTK VNLLSAIKSP CQRETPEGAE AKPWYEPIYL GGVFQLEKGD RLSAEINRPD YLDFAESGQV YFGIIAL
    • Predicted molecular weight

      17 kDa
    • Amino acids

      77 to 233
    • Additional sequence information

      aa 77 to 233 refers to the full length mature form (soluble).

Specifications

Our Abpromise guarantee covers the use of ab9642 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

  • Applications

    Functional Studies

    Sandwich ELISA

    HPLC

    SDS-PAGE

  • Form

    Lyophilised
  • Additional notes

    Lots prior to June 2015 contain 0.036% Tris (including lots GR157466-11, -12 and -13).

  • Concentration information loading...

Preparation and Storage

  • Stability and Storage

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.

    Constituents: 0.049% Sodium phosphate, 0.12% Sodium chloride

    This product is an active protein and may elicit a biological response in vivo, handle with caution.

  • Reconstitution
    Reconstitute with dH2O to make a final concentration between 0.1 to 1.0 mg/ml.

General Info

  • 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
  • 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

Images

  • 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.

  • Chromatin was prepared from HeLa (human epithelial cell line from cervix adenocarcinoma) cells treated with and without 20 ng/ml TNF-α (ab9642) for 60 minutes according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 μg of chromatin, 5 μg of ab218533 (red), and 20 µl of Protein A/G sepharose beads. 5 μg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (SYBR green approach).

    The ChIP data are consistent with the literature (PMID: 16135789).

  • All lanes : Anti-TNFAIP3 antibody [EPR2663] (ab92324) at 1/5000 dilution

    Lane 1 : WEHI-3 (Mouse leukemia lymphoblast) whole cell lysate
    Lane 2 : WEHI-3 treated with 20 ng/ml TNF alpha (ab9642) for 6 h

    Lysates/proteins at 20 µg per lane.

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

    Observed band size: 80 kDa
    why is the actual band size different from the predicted?

  • All lanes : Anti-NF-kB p65 (acetyl K310) antibody [EPR21781] - ChIP Grade (ab218533) at 1/2000 dilution

    Lane 1 : HEK-293 transfected with NF-kB p65 expression vector containing a myc-His-tag®, whole cell lysate
    Lane 2 : HEK-293 transfected with NF-kB p65 and p300 (aa1287-1663) expression vectors containing a myc-His-tag®, then treated with 20 ng/ml TNF-alpha (ab9642) for 60 minutes, 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

    Observed band size: 70 kDa why is the actual band size different from the predicted?


    Exposure time: 37 seconds


    Blocking/Dilution buffer: 5% NFDM/TBST.

    NF-κB p65 (acetyl K310) expression is induced by TNF-α and p300 acetyltransferases (PMID: 20160011, PMID: 12456660, PMID: 16135789).

  • All lanes : Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) at 1/1000 dilution

    Lane 1 : Untreated L-929 (Mouse connective tissue fibroblast cells) whole cell lysate
    Lane 2 : L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642), 100 nM Smac mimetic, and 20 µM z-VAD (ab120382) for 8 h and then harvested.

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

    Observed band size: 54 kDa why is the actual band size different from the predicted?


    Exposure time: 15 seconds


    Blocking and dilution buffer: 5% NFDM/TBST.

  • MLKL (phospho S345) was immunoprecipitated from 1mg of L-929 (Mouse connective tissue fibroblast cells) whole cell lysate treated with 20 ng/ml TNF alpha (ab9642) + 100 nM Smac mimetic + 20 µM z-VAD compound (ab120382) for 8h using ab196436 at 1/150 dilution. Western blot was performed from the immunoprecipitate using ab196436 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

    Lane 1: L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642) + 100 nM Smac mimetic+ 20 µM z-VAD compound (ab120382) for 8h;10 µg (Input).
    Lane 2: ab196436 IP in L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642) + 100 nM Smac mimetic+ 20 µM z-VAD compound (ab120382) for 8h.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab196436 in L-929 whole cell lysate treated with 20 ng/ml TNF alpha (ab9642) + 100 nM Smac mimetic+ 20 µM z-VAD compound (ab120382) for 8h.

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

  • All lanes : Anti-MLKL (phospho S345) antibody [EPR9515(2)] (ab196436) at 1/1000 dilution

    Lane 1 : L-929 treated with 20 ng/ml TNF alpha (ab9642), 100 nM Smac mimetic, and 20 µM z-VAD (ab120382) for 8 h, whole cell lysate
    Lane 2 : Mouse brain tissue lysate
    Lane 3 : Mouse colon tissue lysate
    Lane 4 : Mouse lung tissue lysate
    Lane 5 : Mouse retina tissue lysate
    Lane 6 : Mouse liver tissue lysate
    Lane 7 : Raw264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) 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

    Observed band size: 54 kDa why is the actual band size different from the predicted?


    Exposure time: 50 seconds


    Blocking and diluting buffer: 5% NFDM/TBST.

    MLKL pS345 is a trigger for necroptosis. It is only detectable in infection/cellular damaged (PMID:29229989) or aging tissue (PMID: 28807105) but not in normal tissues.

References

This product has been referenced in:

  • King D  et al. Label-Free Multi Parameter Optical Interrogation of Endothelial Activation in Single Cells using a Lab on a Disc Platform. Sci Rep 9:4157 (2019). Read more (PubMed: 30858536) »
  • Paludo FJ  et al. Effects of 47C allele (rs4880) of the SOD2 gene in the production of intracellular reactive species in peripheral blood mononuclear cells with and without lipopolysaccharides induction. Free Radic Res 48:190-9 (2014). Read more (PubMed: 24164232) »
See all 4 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Answer


The lab let me know that they do not test the half-life/stability of ab9642 at 37oC. However, they have found that in their assay on 929 cells, they incubate this product in culture for approximately 21 hours. It is incubated in assay media containing 2ug/ml of Actinomycin D.

I would thus suggest to test if the protein is still stable after 96 hours, but to be on the safe side a 24 h incubation might be best.

Read More

Question
Answer

Thank you for your enquiry. As I mentioned earlier, these proteins were originated from two different sources outside Abcam. Regarding the specific activity and purity for these antibodies: Ab7420 - Specific activity: 2.34 x 10 to the 6 Units/mg protein. 99% purity. Ab9642 - Specific activity: > 2 x 10 to the 7 units/mg. Greater than 98% pure. Either one should work for you, please take a look again at the online datasheets - all the information that we have is accessible to you. If you have any further questions, please do let me know.

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