Some quantitative aspects of the disc electrophoresis of ovalbumin using amido black 10B stain

The quantitative aspects of the disc electrophoretic technique were investigated using a purified protein, egg ovalbumin. Depending on the filter used, a linear relationship between peak area and protein concentration was found up to about 40 μg of protein by densitometry. Both diffusion and gel slicing studies indicated that linearity could be extended to almost 160 μg of protein. By elution of the amido black dye from the protein-dye complex in the gel, a nearly constant dye to protein ratio was indicated. These results suggested that quantitation of the stained bands on polyacrylamide gels was limited by the nonlinear response of the densitometer, perhaps due to the nonlinearity of dye absorbance at large optical densities and not by variable amounts of dye binding to the protein bands.