Improved method for the determination of hemoglobin A2 by starch-gel electrophoresis; with some observations on the technic.

STARCH GEL as a supporting medium for the electrophoretic separation of proteins, first used by Smithies (8), has found increased application in recent years (3, 4, 5, 10). However, certain difficulties have become evident in the use of this medium: The optimal resolution of protein components was found to depend on the careful selection of the degree and method of hydrolysis of the starch (5, 6), and the reproducibility of the protein patterns on rigid control of the experimental conditions (6). Moreover, the evaluation of starch-gel patterns of proteins by direct photometry requires the use of aqueous staining methods (1) to ensure maximal transparency of the starch gel, or crarification of the starch after the staining procedure (9). Starch-gel electrophoresis has proved to be particularly useful for the determination of hemoglobin A2 (3). This minor component is normally present in concentrations of less than 5 per cent and, even in cases of thalassemia, rarely exceeds 10 per cent of total hemoglobin. These low concentrations require that procedures of the highest possible accuracy be used for its determination. In the initial procedure for starch-gel electrophoresis of human hemoglobins, developed in our laboratory (3), evaluation of the unstained patterns was carried out by densitometry of photographs of these_patterns on Polaroid projection film using reflected light and a