Abstract
Human serum protein fractions were separated by continuous-flow electrophoresis and purified by dialysis, lipid extraction, lyophilization, and drying to constant weight. Increasing amounts of each of the protein fractions were applied to strips of filter paper. One set of strips was stained with amido black 10B dye, and another set with bromphenol blue. The dye-binding properties of the protein fractions were evaluated by (1) the Grassmann-Hannig and (2) Spinco tech nics of direct photometry, and (3) by elution procedures.
The calibration charts indicated that dye-binding is not a strictly linear function of protein concentration. This deviation is especially marked in the range from 0 to 40 µg. of protein. Deviation from the Beer-Lambert law with small quantities of protein leads to artifactitious augmentation in the estimation of proteins present in serum in low concentration, such as the alpha globulins.
In the range from 40 to 80 µg. of protein, with the three methods for quantitation, the affinities of alpha, beta, and gamma globulin for amido black averaged 80, 71, and 82 per cent, respectively, of that of albumin. Under comparable conditions, the affinities of alpha, beta, and gamma globulins for bromphenol blue averaged 64, 58, and 60 per cent, respectively. On the basis of these findings, amido black 10B appears to have a theoretical superiority over bromphenol blue for use in quantitative estimations of serum protein fractions by paper electrophoresis.
Ultramicroanalysis of Serum Protein Fractions by Cellulose Acetate Electrophoresis
