My guess is that you are using an inappropriate standard, namely BSA, for measuring collagen concentration. Collagen has an unusual amino acid composition, quite different from that of BSA. Can you use collagen as the standard?

Adam B Shapiro isn't that a chicken-egg situation? As in, I am trying to quantify the concentration of collagen in a solution of lyophilized collagen type II in acidic solution with a known amount of lyophilized product per mL of solvent. Technically this would be considered a standard. Or am I getting this wrong?

It is a standard only if you know that all mass in the lyophilizate corresponds to protein. This isn't necessarily, and often isn't, the case.

You dissolved a known amount of type II collagen, tried to determine its concentration versus a BSA standard, and the assay did not yield the concentration you expected. Adam B Shapiro comes and tells you that the same amount of different proteins (especially when aminoacid composition is significantly different, as happens to be the case) yields different absorbances in that assay. To me it looks like that might explain your results, doesn't it? Collagen at 1 mg/mL did not yield the same absorbance as BSA at 1 mg/mL, isn't that essentially what you are wondering about?

Hope it helps!

I didn't appreciate that the collagen sample you were trying to quantify was a dry sample. If the collagen you are testing is mixed with other substances, such as salts, then the weight of collagen is less than the weight of sample. As long as the collagen is the only protein present in the dry material, then I suggest dissolving some in water, dialyzing against water thoroughly to remove any low molecular weight contaminants, then freeze drying the dialyzed sample thoroughly to remove all the water. Then you will have pure collagen, and you can just use the weight that you measure with a balance - no protein assay necessary.

If you don't want to take this approach, then try to find a source of pure collagen that you can weigh out and use as a standard for a protein assay to measure the amount of collagen in a sample that contains collagen plus other non-protein material.

Another way to measure the protein content is to have an amino acid analysis performed by a specialized facility.

Adam B Shapiro and Alejandro Martin Thank you very much for your replies. They have been extremely helpful. I followed your suggestion and obtained some commercially available collagen type I of known concentration, made dilutions and performed the assay on know dilutions and compared it to that of BSA. I boiled/denatured all the samples in this graph, I anticipated that the three samples would collapse to one curve. But they didn't. The collagen type II that you see plotted on the graph has been plotted against the expected concentration from the dissolution of the measured lyophilized product.

So, it is safe to assume that the solution of collagen type II in 0.012 M HCl that I am assuming to be 1 mg/ml is indeed.

I am undertaking this exercise to confirm the presence of this peptide in solution and quantitatively obtain a estimate of concentration, as my downstream experiments have been affected possibly by lower concentration.

Thanks again for your inputs, means a lot!

I found the same issue when I tried to estimate concentration of IgY immunoglobulins using BSA as standard. I believe that although proteins solutions have same concentration, it depends on the reagent and its interaction with the particular protein that will result in different absorbance values.

You may find the protein-to-protein variation for collagen relative to BSA. If it is the 40% of expected value, you are not doing wrong.

Carlos Leónidas Leiva the question that i might ask next might seem super lame, but changing the concentration units to mM might help right? Because the moles of amide (the reacting functional group) depends on the molecular weight of the protein.. So would it be right to say that if the protein whose unknown concentration we are measuring is close in molecular wt. to that of the standard, then our estimation would be correct?

One milligram of a 100 kDa protein has the same number of amide grous as one milligram of a 10 kDa protein (barring differences in aminoacid composition) so no, using mM would not help but would rather make things worse (by requiring use of a standard of the same MW as the analyte). Put another way, what you are measuring is mass of amide groups, not the number of protein molecules.

I am writing to confirm that I saw a nearly identical phenomenon with type 1 collagen isolated from rat tail tendons. A commercial solution that I knew should be 8.13 mg/mL (bought from Corning) displayed absorbance values that implied the collagen concentration was actually 3.5 mg/mL, which I knew to be incorrect. Thank you for clarifying my confusion! I will be sure to construct a concentration ladder from the control collagen on my next attempt.