Quantitation of microgram amounts of protein in two-dimensional polyacrylamide gel electrophoresis sample buffer

The concentration of protein actually solubilized in sample buffer in preparation for analysis by two-dimensional polyacrylamide gel electrophoresis cannot be directly determined by the Lowry, Biuret, or Bradford protein methods due to interference by the combinational effect of presence of urea, detergents, carrier ampholytes, and thiol compounds in sample solubilization buffers. Determinations of the actual amount of protein solubilized and applied to gels is required to accurately quantitatively and qualitatively evaluate second-dimension polypeptide maps. It was found that when sample buffer consisting of 9 M urea, 4 % Nonidet P-40, 2 % Ampholines, and 2 % 2-mercaptoethanol containing solubilized sample(s) was acidified prior to dilution, protein concentrations over a range of 0.5 to 50 μg could be reproducibly determined utilizing a modified Bradford assay. The modified assay generates two near-linear segments, one over the range < 0.5 to 5 μg total protein that permits the application of Beer's law and a second linear response encompassing 5 to 50 μg total protein. The assay did not tolerate presence of greater than 0.1 % sodium dodecyl sulfate but addition of sodium chloride and protamine sulfate did not adversely affect protein quantitation. The modified assay allows direct quantitation of protein solubilized in sample buffers containing urea, carrier ampholytes, nonionic detergents, and thiol compounds.