We used BCA Protein Assay to test our MNP conjugate with streptavidin-peroxidase (S5512), however the results showed no change in color of our samples. We don't know why it happened.
The activity of the enzyme itself is unlikely to be a factor because the assay is done at pH 11, at which the enzyme is probably inactive. Moreover, you probably don't have the substrates of the enzyme present during the protein assay.
An excipient in the enzyme preparation could interfere. Here is a list of interfering substances:
https://tools.thermofisher.com/content/sfs/brochures/TR0068-Protein-assay-compatibility.pdf
Sigma product S5512 contains citrate salts at an unspecified amount. The microBCA protocol is compatible with up to 5 mM sodium citrate at pH 4.8, whereas the full-volume BCA assay is compatible with up to 200 mM. Perhaps if you call the technical support at Sigma they will tell you how much citrate is in the product. It is worth mentioning, however, that the product specification sheet mentions using the BCA assay to measure the protein content.
So my guess is that you didn't put enough of the protein into the assay to get a signal.
Run the assay at 60 °C and increase the amount of sampple, avoid the presence of reducing or chelating agents.
Dr. Adam, we both tried using the microBCA and the Pierce BCA kit, both showed no positive results, however, when we tried using our clean MNPs (only for BCA; without streptavidin-peroxidase), we got a signal.
At one point, you might be right that we didn't put enough of the protein into the assay to get a signal. In any case, I will review the list that you gave me, and will discuss the matter to my supervisor. Thank you very much for the detailed explanation. I'll keep you posted if there will be a development.
Dr. Omar, thank you very much for the suggestion, I'll discuss it with my supervisor and we'll see if the temperature might be a factor,
- Badges
- Science method