Urea Assay Kit (ab83362)

We have not tested urea that has been reacted at both amine groups. However, it will likely not work because the enzyme reaction is very specific for urea.

The phenol red could interfere if the amount of medium used is enough to contribute red color to the sample. Typically when diluted medium is used and the volume is made up to 50ul with buffer, the color does not interfere in the assay. Since the probe emits at 570nm and this is the pink-orange-red zone of the spectrum,red color from the medium can interfere if undiluted medium is used.

For all samples, including tissue or cell extracts, add 1-25ul sample to each well and volume up to 50ul with Assay Buffer.

Add 50 ul Urea Reaction Mix to each sample or standard.

Final volume in each well will be 100ul.

To answer your questions:

1) The desired wavelength for reading the samples is 570 nm.

If the detection band width on the plate reader is ± 30, then a wavelength of ± 30 of 570 can be used, which is 540-600 nm. Otherwise the efficiency of detection of this assay will decrease.
However, from an earlier customer inquiry, the lab replied that they would not recommend reading this assay at 590 nm.

This would then also apply to reading the assay at 450 or 620 nm. This would not be recommended as you might not get a signal at these wavelengths.


2) The kit datasheet described what the kit contains (https://www.abcam.com/ab83362.html)

The protocol lists the additional materials that is required as, but not provided:
Microcentrifuge, Pipettes and pipette tips, Fluorescent or colorimetric microplate reader,  96 well plate, Orbital shaker

Thank you for contacting us.

The calculation is right. We suggest in the protocol diluting the standard to get 0.5milli molar per 1000ul.

This 0.5 is a molar solution means, it is 0.5 milli mol per litre per 1000ul i.e. 0.5 nano mol per micro litre.

so 2ul of this solution would be 1nmol/well

4ul would be 2nmol/well and so on.

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 contacting us.

I have double checked your question with the laboratory, and I am happy to confirm, that this kit should work with the buffers your client mentions. It will work with all common lab buffers. The only ones it would not work with are the ones which can inactivate the enzymes in this assay kit. Since we cannot disclose all the components in such mixtures, your client will need to clarify it with you/us before changing the buffers.

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 regarding ab83362.
If the detection band width on the plate reader is ± 30, then a wavelength of ± 30 of 570 can be used, which is 540-600 nm. Otherwise the efficiency of detection of this assay will decrease.
Unfortunately, we not have data showing that hemolysis would affect the reading, but I would think that a cleaner serum sample would give better results.
There is no exact amount of mouse serum which will be right for this assay. For unknown samples, we suggest testing several doses of your sample to ensure the readings are within the standard curve range. I would suggest making a dose curve initially to determine the optimal starting sample volume.
I hope this will be useful for you.

Thank you for your message and for providing this further information.

I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a refund or credit note in compensation.

I look forward to hearing from you with details of how you would like to proceed.