Stable pH Gradient Research Articles

Analytical isoelectric focusing using a high-voltage vertical slab polyacrylamide gel system

A commercially supplied vertical slab electrophoresis system has been modified to permit electrofocusing of thin gels using electrical potentials of 3000 V and higher. Polyacrylamide gels (5.65% T, 2.65% C; 2.4–3.3% ( w v ) ampholytes; 0.35 mm thick × 98–105 mm long × 140 mm wide) were run under native and denaturing conditions. Accurate temperature regulation and atmospheric control were obtained by casting the gel between two glass plates, and then completely submerging the gel in the lower tank buffer. As many as 18 samples were loaded into wells at the top of each gel. Protein standards and mouse ascites fluid were focused on gels in the native state using a broad-range blend of commercial ampholytes from pH 3.5 to 10. Narrow-range pH ampholyte blends were also used: pH 2.5 to 6 under denaturing conditions resolving bovine calmodulins; pH 4 to 6 under a native condition for human plasma proteins including immunoglobulin G, fibronectin, and fibrinogen; pH 4 to 6 under denaturing conditions for myosin light chains; pH 6 to 9 under native conditions for human hemoglobins; and pH 9 to 11 under denaturing conditions to separate 30 S ribosomal subunit proteins. High-voltage vertical slab electrofocusing provides a means for rapid resolution of multiple protein samples using stable pH gradients. The method is especially valuable in ranges near pH 2.5 and pH 10.5 in which difficulties have previously been encountered with regard to atmospheric control and temperature regulation using conventional focusing techniques.

Buffer electrofocusing of Interleukin I.

Buffer electrofocusing, utilizing a mixture of low-molecular-weight buffer compounds to establish a stable linear pH gradient covering the range 3.5-6.0 on granulated gel media, has been employed in the purification of Interleukin I with good recovery of biological activity. The difficulties often experienced in the removal of commercial synthetic ampholytes after conventional preparative electrofocusing are avoided in this method.

Demonstration of a pH gradient across mucus adherent to rabbit gastric mucosa: evidence for a 'mucus-bicarbonate' barrier.

We examined the ability of the mucus layer adherent to isolated pieces of rabbit gastric mucosa to maintain a pH gradient across it. Using antimony microelectrodes, a stable pH gradient was detected from pH 2.31 +/- 0.04 on the luminal side to pH 7.26 +/- 0.15 (n = 22) on the epithelial side of the mucus layer. The gradient was maintained for at least 60 minutes. A metabolic inhibitor, potassium cyanide, markedly reduced the tissues' ability to maintain this pH gradient, suggesting involvement of an active cellular process, probably that of bicarbonate secretion. These observations provide additional evidence in favour of a 'mucus-bicarbonate' barrier which may be of importance in protecting the underlying gastric mucosa.

Generation of stable pH gradients for preparative isoelectric focusing by electrolysis of two‐component buffer solutions in a multi‐compartment apparatus

AbstractA widened use of isoelectric focusing for preparation of large amounts of pure proteins is highly desirable but is severely hampered, inter alia by the high cost of the carrier ampholytes used for creating the pH gradient. Efforts are therefore being made to replace the carrier ampholytes with simple two‐component buffers. To this end, a multimembrane electrolyzer, consisting of 22 compartments with attached cooling loops, has been constructed. The choice of membranes has proved to be one of the main problems. With permeable membranes, even small differences in hydrostatic pressure between the ends of the electrolyzer will cause internal liquid flows that prohibit the formulation of a useful pH gradient. Likewise, flow‐tight membranes generally give rise to a large internal flow due to electroosmosis. Among the types of membranes so far examined, polycrylamide membranes form an exception. With these membranes, useful and stable pH gradients have been created. There are drawbacks, however, relating to the low mechanical strength and to the difficulty of producing exactly equal membranes, thereby fulfilling the theoretical demand of constant transference numbers for the ions. The experimental observation that even small disturbances, such as variations of the transference numbers or a minute electroosmotic flow through the electrolyzer, may completely destroy the pH gradient, has been related to the mechanism operating during electrolysis of a simple buffer. While a possible local breakdown of a carrier‐ampholyte pH gradient is rapidly repaired by the current, a disturbance occurring in a buffer pH gradient can be repaired only by diffusion, which is a slow process.

Isoelectric focusing of immunoglobulins: Improved methodology

Conditions for isoelectric focusing and detecting antibodies in thin layers of polyacrylamide have been evaluated and several improvements have been made. First, we have developed a simple method for covalently attaching the polyacrylamide gel to the glass support which improves the mechanical stability of the gel and removes the need for protein subbed plates. This in turn leads to decreased electroendosmosis and decreased background protein staining. Secondly, we have applied the methods of Nguyen and Chrambach (1977 a,b), in which amino acid solutions are used as electrodes, to focus immunoglobulins. This has eliminated cathodic drift and resulted in extremely stable pH gradients. Finally, we have found that conventional methods for detecting focused antibodies that rely on protein cross-linking before exposure to antigen often lead to distortion of the focusing patterns. Both glutaraldehyde and suberimidate destroy the antigen binding capacity of some antibodies at concentrations too low for complete fixation of protein bands. These fixation artifacts are avoided if salt-precipitated antibodies are exposed to radiolabeled antigen before fixation.

Nonisoelectric focusing in buffers

Stable pH gradients were formed and focusing of proteins was carried out in polyacrylamide gels containing mixtures of simple, amphoteric buffers, replacing the Ampholine hitherto used in isoelectric focusing (IF). Stable pH gradients can also be formed between acid anolyte and basic catholyte if Ampholine is replaced by nonamphoteric buffers. The fact that focusing can be carried out with nonampholytes shows that focusing in this case is, and in all other cases may be, nonisoelectric. It is postulated that the pH gradient in IF forms by steady-state stacking (isotachophoresis) and forms within the stack. In distinction to ordinary steady-state stacking, however, the stack remains confined within the gel (or density gradient) since the strong acid and base in the electrolyte reservoirs bar by deprotonation or electrostatic repulsion migration into the electrode chambers.

Isoelectric focusing of proteins using a pH gradient created by a concentration gradient of nonelectrolytes in solution

A new method of producing a stable pH gradient in buffer solutions is suggested, obtained by the concentration gradient of a nonelectrolyte in buffer solutions as a result of the gradual change in the dielectric properties of the solution. The maximal concentrations of nonelectrolyte which do not influence the protein configuration allow a pH gradient with a range of two pH units to be produced. It is suggested that the properties of some polyols (i.e. glycerol or mannitol) be used to change the pH of the borate buffer for the production of greater pH gradient with a range of up to 4–5 pH units. Creating the gradient of concentration of polyols, one can obtain a pH gradient in borate buffer solutions. Though the polylydroxyl compound-borate complexes possess mobility in an electric field, a stable pH gradient can be achieved during 12 days of electrophoresis. The isoelectric focusing of haemoglobin, human serum albumin and immunoglobulins was carried out in both systems suggested. These findings were compared with isoelectric focusing in Ampholines. There was a good agreement between the methods compared. The possible differences are discussed.

Isoelectric focusing of herpes simplex virus.

Herpes simplex virus was inserted into a preformed stable pH gradient and electrofocused for about one hour. Dextran was used as the density gradient forming agent. Virions banded at pH 4.9+/-0.1 and nucleocapsids at 4.1+/-0.05. About 10-20 per cent of infective virus was recovered.

Fractionation of staphylococcal enterotoxin B by isoelectric focusing

1. 1. In the context of studies concerned with a radioimmunoassay procedure for detection of staphylococcal enterotoxins in food, enterotoxin B was purified from the culture supernatant of Staphylococcus aureus, strain S-6. Purified preparations were subjected to the technique of isoelectric focusing of proteins in a stable pH gradient. The existence of two major electrophoretic forms of the toxin has been confirmed by this method. 2. 2. Both major components as well as a minor component(s) were found to be toxic in monkeys and gave a reaction of immunological identity. 3. 3. In refocusing experiments, the more acidic component of the toxin was found to arise by conversion from the more basic component. 4. 4. The results also indicate the usefulness of zonal electrophoresis in a density gradient for purification of enterotoxin B on a preparative scale.

Isoelectric focusing in polyacrylamide gels

A rapid method for fractionating proteins by isoelectric focusing in small rods of polyacrylamide gel is described. An apparatus is described in which multiple samples can be fractionated in different pH gradients in less than6 h. Smooth and stable pH gradients were achieved by passing a current of 1 mA through high porosity gels containing 2% (w/v) ampholyte with solutions of 0.01 M H 3PO 4 and 0.02 M NaOH as anolyte and catholyte respectively. The pH gradients were highly reproducible, and the desired pH range occupied over 80% of the length of the gel. The advantages and limitations of this technique are discussed and examples are given of its application to the separations of several well-characterised proteins.

A new approach to isoelectric focusing and fractionation of proteins in a pH gradient.

This paper describes a new method of condensation (focusing) of extended volumes of mixtures of proteins (or other ampholytes) into an isoelectric spectrum of discrete zones located at points of a pH gradient corresponding to the pI value of the individual proteins. In contradistinction to the currently practiced isoelectric focusing in "natural" pH gradients which may require as much as 96 hours for completion, the present method yields clear-cut condensations marking the isoelectric pH within about five minutes and complete fractionation within about 15 minutes. No "Ampholines" or other special buffers are required and establishment of the pH gradient requires only about 10 seconds rather than days as is common in natural pH gradient focusing. The pH gradient is generated by utilization of the temperature dependence of pH. By establishing a temperature gradient between 0 degrees and 50 degrees C in an electrophoretic column, a stable pH gradient extending over 1 pH unit can be maintained in the absence as well as the presence of a current. The pH gradient can be easily controlled by variation of the temperature limits so that high resolving power can be achieved in shallow pH gradients. The pI of the focused fractions is determined by measurement (by a thermistor or resistance thermometer) of the local temperature within each of the isoelectric zones that determine the local pH. The individual zones can be pipetted out. The method is illustrated by simultaneous condensation and evacuation of hemoglobin in two pH gradients traversed by opposite currents and by separation of hemoglobins A and S in 16 minutes in a pH gradient where the current passes in the direction of increasing pH in Tris buffer solutions stabilized by a sucrose density gradient.

Studies on extracellular proteins from Staphylococcus aureus: I. Separation and characterization of enzymes and toxins by isoelectric focusing

Staphylococcus aureus, strain V8, was grown in a 4-l liquid culture for 24 h. The culture supernatant was concentrated about 100 times. The crude mixture of extracellular proteins was studied by a method for isoelectric focusing of proteins in stable pH gradients 13, 14. This method permits separation of proteins and determination of their isoelectric points, pI. A great number of proteins were separated, which showed a spectrum of pI's essentially between pH 3 and 11. α-Haemolysin, deoxyribonuclease, staphylokinase, protease and hyaluronate lyase were studied. All of these activities except staphylokinase were found to consist of multiple molecular forms with different pI's. The results obtained are compared with reports in the literature.