A washed carrot substrate, prepared with high yields and easy handling properties, was found to be a suitable substrate for studying cellulolytic and pectinolytic degradation processes. A cellulase from Trichoderma reesei, and Rohament P, a macerating enzyme from Aspergillus alleaceus in endopolygalacturonase, degraded the washed carrot substrate to an extent of 60%. With the combined action of both enzymes, degradation was more than 80%. Simultaneous action of both enzymes was more efficient than their sequential use. The effect of temperature, pH, incubation time, enzyme concentration, and substrate concentration on the degradation by the single enzymes and their mixture were studied. Gas chromatographic sugar analysis revealed that Rohament P liberated glucose, arabinose, and galactose in the low-molecular-weight fraction obtained by ultrafiltration, in addition to high amounts of galacturonic acid. These carbohydrates were also found in the high-molecular-weight fraction (retentate) together with rhamnose and mannose. Cellulase BC released mainly glucose, although galacturonic acid, arabinose, xylose, and mannose were also detected both in the ultrafiltrate and retentate. Morphologically, during Rohament P degradation of the washed carrot substrate, damaged tissues and disintegrated cells were seen, whereas on cellulase BC action mainly disintegrated cell walls were observed.

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

Discontinuous isoelectric focusing in layers of granulated gels is used to separate up to 1000 mg protein with a resolution of 0.01-0.015 PI. High resolution is achieved with (i) gel layers of reduced thickness (1 mm), (ii) high field strength in the final stage of focusing (100-200 V/cm), (iii) long separation distances, and (iv) high Volthour (Vh) products ensuring steady state conditions. We describe approaches to rapid focusing based on either short separation distances or extensive prefocusing with resultant short residence times of the separated substances in the gel. The optimum load capacity is 3-5 mg protein/ml gel-bed volume while 10 mg proteidml gel-bed volume are still well tolerated. At high loading, major componentsare seen in the gel as translucent zones which can be easily identified and harvested. Best results are obtained with Sephadex G-200 (Superfine) or Bio-Gel P-60 (minus 400 mesh). Protein visualization with a new printing technique employing cellulose acetate membranes requires only 2-3 min and is insensitive to protein overloading and adhering gel particles. A simple and efficient method is described for the electrophoretic removal of carrier ampholytes from the focused proteins. High resolution preparative isoelectric focusing should prove attractive for many applications due to its great flexibility with respect to sample size, gel volume, separation time, protein recovery, ease of operation and reasonable price.

A method for protein staining after isoelectric focusing (IEF) in polyacrylamide gel is described in which the total time for fixation, staining and complete gel background destaining is reduced to 3-5 min. This is achieved by (i) staining dry gels on silanized supports instead of hydrated gels, (ii) reducing gel thickness to 50 pm, (iii) using Servalyt carrier ampholytes with good destaining properties, and (iv) using dyes with low affinity for carrier ampholytes, namely Serva Violet 17 or 49, facilitating destaining of gel background. The sensitivity of staining is 100-200 ng protein/ mm2 over a 10 cm separation distance and 20-30 ng protein/ mm2 over 3 cm gels (miniature focusing). Addition of 5-20 % methyl acetate to the staining or destaining solution reduces the sensitivity of staining significantly. Most effective fixation of proteins in 50 pm gels is achieved with 20 % trichloroacetic acid. In solutions of alcohol and acetic acid, appreciable amounts of proteins are leached after incubation for 5-20 min. Paraformaldehyde, malondialdehyde or glutaraldehyde (5-10 %) exhibit no fixative effect on proteins in 50 pm gels when used directly or after precipitation of the proteins with trichloroacetic acid. The selective leaching of proteins under a variety of experimental conditions shows that adequate fixation is particularly important in work with basic, low molecular weight and/or hydrophobic proteins.