Figure 3. Gelatin (a) and casein (b) zymograms showing the relative proteolytic activities in equal volumes of porcine secretory enamel samples obtained at different depths: outer enamel (lane 1), outer-inner enamel (lane 2), and inner enamel (lane 3). The gel of casein zymogram was incubated in 2 mM calcium. The volumes for 100 mg of outer, outer-inner, and inner layer enamel samples were calculated to be 75 µL, 68 µL, and 64 µL, respectively. A 10-µL quantity of each enamel sample was extracted sequentially with 180 µL of Sorensen buffer (pH 7.4) and 0.05 M carbonate buffer (pH 10.8). We prepared samples for electrophoresis by adding the equal volume of 4% SDS/2% sucrose solution to each extracted solution. Equal proportions of each sample volume were applied to the zymograms. For the gelatin and casein zymograms, 20 µL of sample solution prepared from the neutral-soluble fraction and 10 µL of sample solution from the alkaline-soluble fraction, respectively, were applied to each well.