A High Ph Discontinuous Buffer System for Resolution of Isozymes in Starch Gel Electrophoresis

A Tris-citrate pH 9.5 gel/borate pH 8.2 electrode discontinuous buffer system for starch gel electrophoresis of proteins was developed to resolve iso- and allozymes of aspartate aminotransferase in frogs (Hyla crucifer).- This buffer system also enhanced resolution of NADP-dependent malate dehydrogenase and the L-lactate dehydrogenase-A locus in this species. It provided good resolution of NAD-dependent malate dehydrogenase in esocid fishes, and esterases, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phospbate dehydrogenase, alcohol dehydrogenase, and S-aconitate hydratase in ambystomatid salamanders. Variation suppressed by other buffers was revealed by this buffer for some enzyme encoding loci, while at other loci, this buffer suppressed electromorph variability. The concentration of tris(hydroxymethyl)aminomethane in gels made with this buffer was much higher than in pH 8.7 “Poulik” gels, but running characteristics of the two gel types were similar. Gels made with this new buffer were less prone to splitting and “warping” than Poulik gels, and were easier to handle. When screening a given taxon for enzyme variability, tests using multiple buffers are essential to maximize the amount of electrophoretically detectable variation.