Fast and high resolution enzyme visualization in ultrathinlayer isoelectric focusing

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Abstract
Three approaches are described for fast and high resolution enzyme visualization in ultrathin‐layer isoelectric focusing. (i) For direct visualization in the focused gel, solutions with reagent concentrations, increased 10–20 times over those normally used, were applied, accelerating and intensifying color development. Lactate dehydrogenase and alcohol dehydrogenase were visualized by overlayering the focused gel with the substrate and reagent solution. (ii) Membrane printing, employing polyamide or cellulose acetate membranes, pretreated with buffer and impregnated with the staining solution, was superior to direct visualization for some enzymes. Glycosidases were located by salting‐out in the focused gel with 90% ammonium sulfate, followed by printing with membranes impregnated with 4‐methylumbelliferyl derivatives of carbohydrates. (iii) Ultrathin (100–200 μm) agarose gels, equilibrated before use with solutions containing either low or high molecular weight substrates, buffers and other reagents, replace the traditional overlay technique. Proteases (Pronase P) and phosphoglucomutase were visualized with ultrathin replicas. With all approaches visualization was completed within 2–5 min. For some enzymes elevated temperatures in the range of 60–80°C accelerated visualization and drying of the gel. With all techniques a permanent document was obtained which could be evaluated densitometrically and conveniently stored for later reference. The combination of high resolution separation and detection, capable of handling up to hundreds of samples within a fraction of an hour, and a minimum of effort and costs, is an attractive option for many potential applications.