Quantitation of amylases in Drosophila separated by acrylamide gel electrophoresis

A technique was developed for the qualitative and quantitative analysis of amylases separated by disc electrophoresis. Gels in which separation occurred were incubated against a starch-acrylamide film attached to a glass plate. This was subsequently stained with iodine, covered with another plate, and scanned with a densitometer. Enzyme activity as well as an impression of the gels were transferred to the film, thus providing a permanent record of both banding patterns and activity in each band without destroying the gels. A direct relationship was demonstrated between enzyme concentration and change in optical density of the substrate in the bands on the stained film. It was also shown that the amylases remaining in the gels were fully active following electrophoresis and incubation. They could then be cut out, eluted and used for further analysis. The method was applied to extracts from individual adults of eight Amy strains of D. melanogaster, each characterized by a different amylase banding pattern. Patterns formed by separation in acrylamide gel were compared with those previously described for agar gel electrophoresis. Relative activity in each band of a given pattern was determined in addition to the actual activity, which was calculated in mg starch hydrolyzed/minute. Total activity, or the sum of the activities in all bands of a given genotypic pattern, was calculated for each fly. The results fell within the range of expected variability expressed by single flies of each genotype when subjected to standard methods of starch-iodine assay.