Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients.

An optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5% T, 3% C, were internally stabilized with the aid of an AcrylAide-pretreated, hydrophilized polyester fabric, preferably with a 60 microns mesh opening. Ultrathin (120-180 microns) gels were prepared with the flap technique and 500 microns gels with the cassette technique; 500 microns gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1-3% w/v carrier ampholytes. Incorporation of 40-60% w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900-1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1-0.2 micron pore size nitrocellulose membranes in 10-20 min from ultrathin gels and in 30-60 min from 500 microns gels. Based on quantification of residual protein in the gels after blotting, a transfer efficiency of 60-87% was found for the ultrathin and 53-69% for the 500 microns gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris-glycine buffer system electrophoretic blotting required only 2-5 min with ultrathin gels and 20 min with 500 microns gels. Marker proteins, including horse spleen ferritin (Mr465,000), could be transferred with 91-96% efficiency.

Fast and sensitive protein staining with colloidal acid violet 17 following isoelectric focusing in carrier ampholyte generated and immobilized pH gradients.

A new method is described for fast and sensitive staining of proteins following isoelectric focusing in carrier ampholyte and immobilized pH gradient polyacrylamide gels. After fixation with trichloroacetic acid the gels are stained for 5-10 min with 0.1-0.2% colloidal Serva Violet 17 (generic name: Acid Violet 17; Color Index No. 42,650) in 10% w/v phosphoric acid. After staining for only 0.5-3 min, major zones, corresponding to 100-500 ng protein, are visible without destaining on a weak background. Detection of minor components requires destaining with 3% w/v phosphoric acid for 5-80 min depending on gel thickness (120-500 microns) and type of support (fabric reinforced versus gels backed to a polyester film). For selected pH marker proteins (bovine serum albumin, carbonic anhydrase, horse myoglobin) a staining sensitivity of 1-2 ng/mm2 protein is found. Dye elution from stained fabric reinforced gels with 50% v/v dioxane-water, followed by absorbance measurements, results in a linear relationship over a range of 1-100 micrograms marker proteins. Staining with collodial Serva Violet 17 is the only method available for fast and high sensitivity and low background staining of immobilized pH gradient gels, without interference from selective dye binding in different pH ranges. Staining with the collodial dye is convenient by avoiding organic solvents with unpleasant vapors and potentially hazardous.

Proteins and peroxidase in callus and suspension cultures of apple : a study using ultrathin-layer isoelectric focusing, sensitive silver staining of proteins, and peroxidase isozyme visualization.

Different methods for the isolation of soluble proteins were applied to cell cultures of three apple cultivars (Malus 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.

Degradation of a washed carrot preparation by cellulases and pectinases.

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.

Structural damage of gamma-irradiated ribonuclease revealed by thin-layer isoelectric focusing.

Ribonuclease, irradiated with 60Co gamma-rays in dilute aqueous solution or in the dry state, has been investigated with respect to its charge and size properties. Thin-layer isoelectric focusing revealed extensive change in irradiated RNase; new enzymatically-active components, mainly with isoelectric points lower than in unirradiated RNase were observed. Thin-layer gel chromatography indicated the formation of aggregates which are partially active enzymatically. Aggreation depended on enzyme concentration and was less in more dilute solutions-- at equal degrees of inactivation. Structural damage in the so-called 'native' monomers was revealed by thin-layer isoelectric focusing, their charge properties being distinctly modified.

Isoelectric focusing in layers of granulated gels. II. Preparative isoelectric focusing.

A method for preparative isoelectric focusing of 0.1-10 g amounts of proteins is described. For anticonvective stabilization of the pH gradient, layers of granulated gels (E.G. Sephadex or Bio-Gel) of variable length, width and thickness were used either on glass plates or in troughs. Load capacity, defined as the amount of protein per ml gel suspension, was determined to be 5-10 mg per ml for total protein, irrespective of the pH range of the carrier ampholytes. For single proteins load capacities of 0.25-1 mg per ml were found for pH 3-10 carrier ampholytes, and 2-4 mg per ml for narrow pH range ampholytes. Experiments on a quartz plate followed by densitometric evaluation in situ at 280 nm have demonstrated that it is possible to proceed from analytical thin-layer isoelectric focusing to preparative separations without loss of resolution, just by changing the dimension of the gel layer and increasing the protein load. Improved resolution which facilitates isolation of isoelectrically homegenious components could be achieved on a 40 cm long separation distance. The geometry of a layer is favourable to heat dissipation and this permits the use of high voltage gradients. Recovery of the focused proteins is high an elution simple. The efficiency of the method is illustrated by examples showing separations of single proteins and protein mixtures.

Fractionation of horseradish peroxidase by preparative isoelectric focusing, gel chromatography and ion-exchange chromatography.

Horseradish peroxidase has been fractionated by preparative isoelectric focusing in a density gradient and in a layer of granulated gel using pH-3-10 and narrow-pH-range carrier ampholytes at different total enzyme loads. The resolution of peroxidase isoenzymes in preparative-layer isoelectric focusing was comparable to that obtained by analytical thin-layer isoelectric focusing. Isoelectrically homogeneous isoenzymes could be isolated with good recovery in a single fractionation step. Despite the excellent separation of the individual isoenzymes by isoelectric focusing in gel layers, an effective purification, indicated by the absorbance ratio A403mn/A278nm, could not be achieved by focusing applied as a single step. By different fractionation sequences combining gel chromatography, ion-exchange chromatography, and isoelectric focusing, individual isoenzymes with a high purity and homogeneous with respect to their size and charge properties have been isolated.

Isoelectric patterns of peroxidase isoenzymes from tobacco tissue cultures.

Peroxidases from tobacco tissue cultures have been separated by thin-layer isoelectric focusing into 12-14 isoenzymes, which have been divided into three groups according to differences in isoelectric points. The isoelectric patterns of callus tissues with and without buds have been compared with those of leaves and stems developed in vitro. Qualitatively, there was a basic similarity of the isoelectric patterns, the same isoenzymes being present in all samples. Distinct quantitative differences in the content and substrate specificity were noted for some of the isoenzymes.