"disappeared" Proteins Using Bradford - Bradford reaction does not give correct results (Sep/14/2004 )

Dear all,

I used the Bio-Rad Bradford reagens to determine the protein content in mussel samples (Mytilus edulis). The samples were first freeze-dried and grinded, and then resuspended in either pure water or Tris/HCl-buffer (10 mM Tris / 85 mM NaCl, brought on pH 7.4 with HCl)

After checking my results, it turned out that only 25%-30% of my total dry weight were proteins, while in literature values of 40-70% are given. I fear that part of my proteins were not resuspended, and therefor not measured with the Bradford assay

Did somebody else experience this problem?
Does anybody knows a good protocol for determination of insoluble proteins, or for resuspending proteins in a way Bradford can be used?

Kind regards

hi

bradford's assay will react with the basic aminoacids (e.g. arginine) mostly, so if there are nor many arginines in your protein, there's an error in the measured value.

the other thing is that you get about 25-30 % SOLUBLE proteins in your fraction, so all lipophilic proteins will not be in your aquaneous solution. maybe they are the missing 10%? good solubility is achieved by resuspendig in 8M urea, but then the proteins are not functional anymore, of course. and urea itself reacts with bradford reagent, so be sure to measure against the proper negative control!

mike