Mouse TNF alpha ELISA Kit (ab100747)

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.

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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.

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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.

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.

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.

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!

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.

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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.

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.