Once the proteins have been separated on a gel, a set of staining protocols must be followed to reveal the isozymes. Specific enzyme activity staining is what makes isozyme analysis possible. Recipes and techniques for staining hundreds of enzyme activities are provided in numerous compilations (Micales et al. 1986; Selander et al. 1971; Shaw and Prasad 1970; Soltis et al. 1983) and are beyond the purview of this chapter. However, a brief description of the major types of staining systems is provided here. Three staining systems are used commonly for isozyme analysis: positive, negative, and fluorescent. The vast majority of isozyme visualization systems are based on positive staining. The general idea is that activity of a specific enzyme generates a colored precipitate at zones of enzyme activity. These isozymes are visualized as dark bands against a light background (Figure 1). Negatively stained isozymes are visible as light-colored bands against a dark gel. Common negative staining systems include those for the enzymes superoxide dismutase and catalase. A third class of enzyme staining system works by generating bands that fluoresce when exposed to ultraviolet light. Commonly used fluorescent staining systems include those for arylesterase and b-glucosidase.
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