Overview

  • Product name

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

    Colorimetric
  • Sample type

    Cell culture supernatant, Plasma
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    < 60 pg/ml
  • Range

    93.75 pg/ml - 6000 pg/ml
  • Recovery

    93 %

    Sample specific recovery
    Sample type Average % Range
    Cell culture supernatant 92.46 84% - 104%
    Plasma 93.82 83% - 102%

  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Mouse
  • Product overview

    Abcam’s TNF alpha Mouse ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Mouse TNF alpha in plasma and cell culture supernatants.


    This assay employs an antibody specific for Mouse TNF alpha coated on a 96-well plate. Standards and samples are pipetted into the wells and TNF alpha present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Mouse TNF alpha antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of TNF alpha bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.


    Mouse TNFa levels are extremely low, and as such we expect “normal” levels to be undetectable when using our kit on plasma samples.


    We have not been able to detect endogenous Mouse TNF alpha in normal serum with ab100747, only in serum spiked with Mouse TNF alpha.

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Microplate

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 1 x 96 tests
    20X Wash Buffer Concentrate 1 x 25ml
    300X HRP-Streptavidin Concentrate 1 x 200µl
    5X Assay Diluent B 1 x 15ml
    Assay Diluent A 1 x 30ml
    Biotinylated anti-Mouse TNF alpha (lyophilized) 2 vials
    Recombinant Mouse TNF alpha Standard (lyophilized) 2 vials
    Stop Solution 1 x 8ml
    TMB One-Step Substrate Reagent 1 x 12ml
    TNF alpha Microplate (12 x 8 wells) 1 unit
  • 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

Applications

Our Abpromise guarantee covers the use of ab100747 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

  • TNF-alpha was measured in cell culture medium from Raw control cells, or cells stimulated with PMA (10 ng x mL-1; 24 h) and LPS (1 ug x mL-1; 6 h). Results are shown with background signal subtracted (duplicates +/- SD).

  • Representative Standard Curve using ab100747

  • Representative Standard Curve using ab100747

Protocols

References

This product has been referenced in:

  • Chang RC  et al. Preconception paternal alcohol exposure exerts sex-specific effects on offspring growth and long-term metabolic programming. Epigenetics Chromatin 12:9 (2019). Read more (PubMed: 30670059) »
  • Xia YF  et al. Non-canonical Wnt signaling contributes to ventilator-induced lung injury through upregulation of WISP1 expression. Int J Mol Med 43:1217-1228 (2019). Read more (PubMed: 30664165) »
See all 37 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Answer

The ELISA assays are compatible with RIPA buffer. However, you should prepare your standards and positive controls in the same buffer.

The Bradford assay is compatible with RIPA buffer if the samples are diluted at least 10-fold with distilled water. Thermo Scientific/Pierce has a useful guide to protein assays in PDF form at the following link. http://www.piercenet.com/files/1602063_PAssayHB_122910.pdf . On page 12-13 you will see a table that compares the compatibility of various protein assays with many reagents, including RIPA buffer. The table refers to the Bradford assay as "Coomassie", an alternative name. The BCA assay is considered to be a better choice than the Bradford assay for assays of samples that contain detergents, but if you dilute your samples before assaying them, the Bradford may be adequate.

Read More

Answer

Thank you for contacting us.


Although this kit has not been tested on lysates, it will likely work well using at least a 5 fold dilution of Assay Diluent B.

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

Read More

Answer

Thank you for contacting us.

Unfortunately, this kit’s cross reactivity with human has not been determined. If it helps, it looks like the homology of the extracellular domain between human and mouse is only around 70-77% which is not significantly high and both antibodies used are monoclonal so the kit is quite specific for mouse.

The only simple way to know for sure would be to spike in only human TNF alpha and see if the assay detects it. I wish I could give a more definite answer on this one.

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
Answer

Thank you for your enquiry which has been forwarded to the scientific support team.

I can confirm that these three ELISA kits ab108870 ab100747 and ab100697 have been tested on mouse plasma and serum samples. (We would not recommend to test whole blood).

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

Read More

Answer

Thank you very much for your email and for sending your absorbance values.

I've reviewed this data with our lab, and we do agree that these standard absorbances are weaker than expected. Did yourun any samples as well?This would help narrow down whether the issue is with just the standard protein only or with a common detection reagent.

All of the kits that you've used are from the same production batch, so we wouldn't expect much inherent differences between the kits, but if you think there is something wrong with an individual component we can certainly replace the kit again for you.

I’ve attached some tips from the lab regarding how to get strong signal from the standards, and also forimproving inter-assay variability which might be helpful. Some of the more useful points:

Once reconstituted (50ng/ml), the standard should be stored at ≤-20 for no more than 1-2 weeks. An extra vial of Item C is included in every kit to limit storage issues.
All other reagents once thawed need to be stored at 4°C for no more than 1 month. Do not store any reagent in their diluted form, they are not as stable diluted.
Do not vortex the standard to mix. Rather gently mix by pipetting the reconstituted standard solution up and down several times with a pipette.
Briefly centrifuge reagent vials prior to opening to ensure complete recovery of the lyophilized powder or liquid concentrate as power/liquid can adhere to the inside walls or cap during transit.



It isn't necessary to block the pre-coated plate, it is ready to use. Generally these prepared plates are already blocked afterthe capture antibody is coatedto the wells. The stop solution is sulfuric acid, which is commonly used to stop the reaction that is occuring between the HRP andTMB. Before sulfuric acid is added, the reaction mixture is producing a cation free radical that is blue, but adding the acid results in a diimine terminal product (yellow) that is more stable than the blue radical.

Iapologize if I'mmisunderstanding the question about LPS hydrolysis, but based on a search of the literature it does look like sulfuric acid itself can cause LPS hydrolysis-

http://www.biochemj.org/bj/126/0395/1260395.pdf

I hope that this information will be useful, and I look forward to hearing from you. Please let me know if you have any further questions or if there is anything else that we can do for you.

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Question
Answer

Thank you very much for your call today and for letting us know about the trouble with this new kit.

As we discussed, I'm sending a free of charge replacement kit on the order ***, which I expect to arrive Tuesday or Wednesday of next week. Please keep me updated about the results with this new kit. If you have any standard curve data to send, it would be very helpful so that we can further investigate the problem with this kit.

Please let me know if you have any questions or if there is anything else that we can do for you. Have a nice day!

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Abreview for TNF alpha Mouse ELISA Kit

Good Good 4/5 (Ease of Use)
Abreviews

Sample: Mouse Cell (Homogenised lung tissue)
Specification: Homogenised lung tissue
Amount of Sample: 100 µg
Type of Kit Used: ELISA
Positive control: Virally infected lung tissue.
Did you use the Abcam Kit protocol?: Yes

Additional data
Additional Notes: Overall, a good kit. However, we struggled to detect TNFa at the highest concentration (6000pg/ml) due to too much signal when analysing at 450nm.

Tissue samples (5mg/ml) can be diluted between 1/5-1/100 and attain useful results.

Abcam user community

Verified customer

Submitted Aug 17 2012

Answer

Thank you for contacting us.

The following ELISA kits for mouse ICAM, VCAM, IL-6 and TNF-alpha are suitable for mouse samples:

ab100688 (https://www.abcam.com/ICAM1-Mouse-ELISA-Kit-1-x-96-Well-Plate-ab100688.html)
ab100713 (https://www.abcam.com/IL6-Mouse-ELISA-Kit-1-x-96-Well-Plate-ab100713.html) was tested on tissue as well.
ab100747 (https://www.abcam.com/TNF-alpha-Mouse-ELISA-Kit-1-x-96-Well-Plate-ab100747.html)
ab100750 (https://www.abcam.com/VCAM1-Mouse-ELISA-Kit-1-x-96-Well-Plate-ab100750.html)

Please let me know if these are kits you were refering to.

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

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for getting back to me and for sending us your data. I will forward this data and your feedback onto the lab.

Your credit note ID is ***.

I again apologize that thiskit did not perform as stated on the datasheet. As you have paid by credit card, the purchase price (including shipping and taxes) will be refunded to your card as soon as our accounting department approves.

If you have any questions about this refund, our accounting department can be contacted by email at us.credits@abcam.com or by telephone at 888-772-2226. Please refer to the credit note ID in any correspondence with our accounting department.

Please let me know if there is anything else that we can do for you in the future, and I wish you the best of the luck with your research.

Read More

Answer

Thank you for contacting me about this, and I am sorry to hear that the results were not as expected. I believe that the values given in my previous email were for normal plasma samples, but I will email the lab to check on that.

Could you just send a brief overview of how the plasma samples were prepared? Were the standards diluted in the assay diluent A as well? Could you send a file of your data, as we might be able to suggest some steps to optimize the results?

If the samples and standards were diluted in diluent A, and the sample values are lower than the standards, then there may be some matrix effect from the sample and youcan try a couple of different dilutions to see if this effect can be minimized.

I understand that your samples are limited, and though it may be possible to optimize these results, we do guarantee the kit to work and Ican certainlyarrange a replacement, credit, or refund if you prefer.

Please let me know if you have any questions or if there is anything else that we can do for you, and I will be happy to help.

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1-10 of 11 Abreviews or Q&A

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