Dear Researchers,
I have recently encountered with BCA peptide assay after Trypsin digestion. I have to quantify the 35ng/uL of protein after digestion of 35ng/uL of BSA. After several times, I realized that I found the same absorbance of Blank and this concentration of peptides. Also, blank sample passing through all the steps of digestion and standard blanks has different absorbance. I did reduction, alkylation, digestion, and desalting. However, I have found blank and 35ng/uL concentrations have the same absorbances. Moreover, I tried with other concentrations of protein like 25 and 15ng/uL, and I found the same absorbance.
I would be glad if someone could give me the answer. Is this because of the small amount of protein here?
Does your BCA assay detect the35 ng/uL of BSA that hasn't been digested. In other words, is it just an assay sensitivity issue? You may be able to improve the sensitivity by changing the assay volumes. Have a look at this chart:
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/protein-assays/bca-protein-assays.html
Adam B Shapiro based on the BCA peptide assay standard curve, LOD and LOQ are at this level. I did this in 384 plates but was unable to quantify the relevant amounts. In addition, I used peptide assay and was able to get the standard curve of the appropriate range. However, after digestion in my sample, all the absorbances are the same. Even blank control of the trypsin digestion without protein has a higher absorbance than only the buffer. I used desalting steps, but still unable to get rid of this. Does this make sense? Hoping for the suggestion!
I think you probably lost all the protein to surface adsorption and in the desalting column (if that's what you used), due to the low concentration.
It would be better to start with a much more concentrated protein solution.
Adam B Shapiro greatly appreciated your suggestion. I will mention my experiment later. Have a wonderful time!
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