Ultrathin-layer isoelectric focusing in polyacrylamide gels on cellophane.

A method of ultrathin-layer isoelectric focusing in 0.12-, 0.24-, or 0.36-mm polyacrylamide gel layers polymerized on a sheet of cellophane as support is deseribed. The gel adheres firmly to the cellophane during all operation steps, is protected from fracture, and can be handied very conveniently. Resolution is markedly improved in ultrathin gels in comparison with the conventional 1- to 2-mm-thick gels. Staining and destaining are completed in a substantially shorter time than so far achieved. The ultrathin gels can be easily dried on the cellophane, a perfectly transparent record being obtained for future reference and for densitometric evaluation. Results are presented for a number of commercial proteins and legume seed proteins. The advantages of ultrathin-layer isoelectric focusing are discussed.     

Peptide analysis by isoelectric focusing in polyacrylamide gels

We have examined the use of isoelectric focusing in polyacrylamide gels for the analysis of heterogeneous mixtures of cyanogen bromide peptides. High resolution, sensitivity, and reproducibility are obtainable under conditions which are described. Peptides having molecular weights above 1000 or 2000 can be visualized by fixation and staining. The presence of urea in the gels is important to the procedure; formation of carbamylated derivatives from this cause occurs at most in trace amounts in unfavorable cases. No artifactual heterogenelty from any other cause was apparent.     

Scanning isoelectric focusing of cholera enterotoxin in polyacrylamide gels

Scanning isoelectric focusing has been employed for continuous monitoring of the isoelectric spectrum of highly purified cholera enterotoxin in 4% polyacrylamide gels containing 2% ampholytes pH 3–10. The resolution obtained by this technique is of high order because at no instance during focusing interruption of current occurs and thus diffusion of the isolated protein moieties is suppressed. An added aspect of scanning isoelectric focusing was that it allowed estimation of the minimal focusing time of cholera enterotoxin. Thus under the standard assay procedure, the main basic component of cholera enterotoxin was focused in 5800 sec, while the other at least 3 minor acidic and anodic components were focused in approximately 19000 sec. Focusing of cholera enterotoxin in the presence of 6m urea allowed the visualization of 5 well defined and about equal components. The proteinaceous nature of the observed peaks was verified by scanning at wavelengths other than 280 nm, staining of gels for protein, and varying the concentration of the enterotoxin. The design of scanning isoelectric focusing equipment is presented. Reproducibility, economy of sample, and ampholytes and simplicity of experimental technique were some of the features of this apparatus. The resolution of scanning isoelectric focusing was found to be superior to that of ordinary disc and SDS gel electrophoresis.     

Segregation of membrane components using isoelectric focusing in polyacrylamide gels

A method for efficiently fractionating human erythrocyte membranes into their individual components is presented. This procedure employs isoelectric focusing in polyacrylamide gels after solubilization of the membranes in 8M urea, 0.02M EDTA, 0.2% 2-Me. Electrofocusing of membranes affords higher resolution than previous membrane separation procedures. Also, this method is rapid, requiring 72 hours at most to separate, stain and destain the membrane components in the polyacrylamide gels.     

Transient state isoelectric focusing: theory

Mathematical theory is developed to describe the transient state of isoelectric focusing (pH-gradient electrophoresis) in a linear pH gradient under highly idealized conditions. This theory makes it possible to predict the concentration profile (distribution) for the protein or other amphoteric species of interest as a function of time, when the sample is applied in a zone of infinitesimal thickness at one end of the column, or in a uniform distribution throughout the column. Further, the position of the centroid, and the second moment around the mean, σ², (square of the standard deviation of peak width) are described as a function of time, irrespective of the initial distribution of the protein in the column. Three arbitrary stages of the “focusing” experiment are considered: (1) Focusing, wherein the sample is applied to a preformed pH gradient; (2) Defocusing, which occurs when the electrical field is abolished after an arbitrary time (usually after the concentration profile has begun to approach its steady state) and diffusion is allowed to occur. (3) Refocusing, which occurs after the electrical field is reapplied. Although stages 1 and 3 are conceptually identical aside from the difference in initial conditions, they may differ in several important respects in practice, both with regard to technical problems of measurement, and with regard to the closeness of conditions to the stated assumptions.This theory should make it possible to predict the time necessary to achieve any desired degree of focusing, i.e., approach to the steady-state distribution. Further, this theory and the techniques of analytical scanning isoelectric focusing provide the basis for measurement of the apparent diffusion coefficient (D), the derivative of velocity with respect to position, and if the field strength is known, the slope of the mobility-pH curve at the isoelectric point, {$\text{dM}\text{d(}\text{pH}\text{)}$}.     

Transient state isoelectric focusing. Resolving power and zone resolution

A digital on-line data acquisition system has been used in conjunction with a scanning isoelectric focusing assembly for the direct calculation of the resolving power and zone resolution in transient state isoelectric focusing experiments. The application of the method to the evaluation of these two parameters in relation to (a) the applied electric field strength during focusing, and (b) to the diffusion in the absence of current is presented. A linear relationship between $\text{1}\text{σ}{}^2$ (where σ is the standard deviation of the concentration distribution of a focused zone) and the applied voltage was demonstrated. The resolving power and resolution were generally improved with increased electric field strength.     

Formaldehyde fixation of proteins and small polypeptides after isoelectric focusing in polyacrylamide gels

High-sensitivity detection of proteins and small polypeptides after isoelectric focusing is achieved by immersing the polyacrylamide gel directly in an aqueous Coomassie brilliant blue G-250 solution containing 40% methanol and 4% formaldehyde. The carrier ampholytes remain soluble under these conditions, whereas polypeptides over a wide range of molecular masses and isoelectric points are precipitated within the gel matrix.     

Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.     

Fixation and immunoperoxidase staining of oligopeptides after isoelectric focusing in thin polyacrylamide slab gels

In this paper a new method for immunological detection of small peptides after isoelectric focusing in thin (200 micron) polyacrylamide slab gels is presented. The peptides are immobilized immediately after focusing by pressing a sheet of glutaraldehyde-impregnated filter paper onto the gel. By this procedure, the gel adheres to the paper and this press-blot can be stained using immunoperoxidase staining procedures. Using the two-step peroxidase conjugate and the three-step peroxidase-antiperoxidase method, several oligopeptides could be visualized after focusing. The detection limit of this method appears to be in the nanogram range.     

Direct tissue isoelectric focusing on ultrathin polyacrylamide gels. Applications in enzyme, lectin and immunohistochemistry

Summary Application of cryostal sections directly onto ultrathin polyacrylamide gels and subsequent isoelectric focusing allows elution of proteins, glycoproteins and peptides out of the sections into the gels. The eluted compounds reveal clearly delineated band patterns in the polyacrylamide gels. The advantage of this method is that enzyme histochemical reactions can be directly performed in the gel and in the electroeluted tissue sections. Therefore, this method is suitable for specifying, in more detail, histochemical enzyme reactions and for detecting multiple forms of enzymes even from a single tissue section. Furthermore, the transfer of proteins, glycoproteins and peptides from the gel onto nitrocellulose by a modified Western blot procedure offers the possibility of checking findings obtained by lectin histochemistry and immunohistochemistry.     

A simple sensitive method, applicable to small polypeptides, for the determination of proteins in polyacrylamide gels after isoelectric focusing.

A simple method is described for the determination of polypeptides and proteins in polyacrylamide gels after isoelectric focusing. Precipitates formed in trichloroacetic acid, under controlled conditions, are quantified densitometrically by measuring the proportion of light scattered. The procedure is of particular value in its applicability to smaller polypeptides, with mol.wts. of 3000-6000, which are not fixed adequately in gels by other procedures currently in use. The working range, over which polypeptide concentration is proportional to the effective absorbance, is approx. 1-30 microgram per component.     

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.     

Improved typing of human serum transferrin by isoelectric focusing on ultrathin layer polyacrylamide slab gels

Summary An improved method for separating transferrin subtypes through the use of isoelectric focusing on ultrathin layer polyacrylamide gels is described. The most considerable problems encountered in the assessment of the Tf phenotypes after pretreatment with iron donor compounds are pointed out. Useful technical devices for reducing the occurrence of artifactitious extrabands are suggested. Finally, the gene frequencies in three samples from Italian populations are reported.