ABSTRAC1Different methods for the isolation of soluble proteins were applied to cell cultures of three apple cultivars (Maims sylvestris Mill.), best results being obtained with a rapid technique based on freezing and thawing. Ultrathin-layer isoelectric focusing followed by an improved silver staining method has shown that proteins from apple callus cultures consist of some 60 to 80 zones, with isoelectric points mainly between pH 4 to 7. Depending on protein content, adequate silver staining is achieved with 50 to 500 cells. Protein patterns of callus cultures allowed clear discrimination of cultivars. Protein and peroxidase isozyme patterns in cell saps of suspension cultures show striking differences during the growth cycle, whereas the protein patterns from the nutrient media were constant over the entire cultivation period and closely resembled the patterns of stationary phase and callus cells.

A new approach to isoelectric focusing in polyacrylamide gels is described, based on the use of rehydratable gels which in dry form could be stored for extended periods and which prior to use were rehydrated with solutions of any composition. Ultrathin 60-240 pm polyacrylamide gels, with different composition (5 % T, 3 % C; 3 % T, 4 % C ; 3 % T, 20 % C), were polymerized under well-standardized conditions and, after polymerization, washed exhaustively with distilled water to remove any unreacted monomers, catalysts or solublepolymers. The washed gels wereimpregnated with suitable additives, before drying, to preserve gel functionality on storage. Polyol compounds, such as glycerol, sorbitol and dextran, as well as synthetic polymers like polyethylene glycol and polyvinylpyrrolidone, were the most efticient additives when incorporated into the gel in a concentration of 1 -10 %, either as single substances or in different combinations. Prior to isoelectric focusing the dry gels were rehydrated to the original gel volume with a solution of carrier ampholytes, in some experiments with added separators or urea. Kinetic studies have shown rehydration, depending on gel thickness, to be complete within a few minutes. Isoelectric focusing in rehydratable gels was consistently more reproducible than in wet gels by such criteria as regularity of patterns and coalescence of marker proteins, including ferritin, applied at different positions. Rehydratable gels tolerated higher field strengths at the final stage of isoelectric focusing, with typical valuesof 500-900 V/cm and thus, at agiven volt x hour product, equilibrium focusing could be attained in a shorter time, with improved resolution and sharper zones. Ultrathin-layer isoelectric focusing in rehydratable gels proved insensitive to high salt concentrations (up to 0.5 M) of the samples. Rehydratable gels represent a new generation of gels, excelling over the traditional wet gels by better standardized properties, convenient handling and unsurpassed flexibility. tetramethylethylenediamine; DHEBA, NJ-( 1,2-dihydroxyIethylene)bisacrylamide; HEPES, N-(2 hydroxyethyl)piperazine-N'-2 ethanesulfonic acid; ACES, N-(2-acetamido)-2-aminoethanesulfonic acid; BICINE, N.N-bisl2-hydroxyethyl)glycine; Vh, volt X hour; IPG. immmobilized pH gradient 'D

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 pm) 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 O 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 afractionof an hour, and aminimum ofeffort and costs. is an attractive option for many potential applications.