Gradient Electrofocusing

The technique of isoelectric focusing requires apparatus of very simple design. The principle assumes that a steady state has been reached. This means that no attention need be paid to protein zones moving through the column, nor to the distance between electrodes and the main compartment where separation takes place. Nor are other special devices needed to prevent acidic or basic products of electrolysis from penetrating the separation compartment. In other words, the electrodes can communicate directly with the separation compartment. 

The carrier ampholytes adjacent to the electrodes must be protected from anodic oxidation or cathodic reduction. This is done by using special electrode solutions, which perform the function of a liquid lock. A dilute solution of an acid such as phosphoric or sulfuric acid is used at the anode. A dilute alkali such as sodium hydroxide is used at the cathode. Electrolysis attracts the acid and the base to the respective electrodes. The acid at the anode gives a positive charge to the carrier ampholytes there. Thus they are repelled from the anode. At the cathode, the base gives a negative charge to the carrier ampholytes nearby. They are then repelled from the cathode. 

After the completion of the experiment, the solution must be pumped out of the column or manually drained to prevent the focused zones from remixing. The experimenter will also desire to analyze the solution after withdrawal and to be able to chart each vertical interval of the column with respect to the data desired. 

In the first place, the isoelectric point of the protein should be established, together with its position along the vertical, longitudinal axis of the column. This is often done by localizing the protein zones by measur- 

LKB-Produkter AB, Stockholm, Sweden, has introduced electrofocusing by making available supplies of carrier ampholytes and columns for electrofocusing. This is done under the trade name of Ampholine. There are two kinds of column. One is specially designed to meet the 

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Isoelectric focusing and electrofocusing are names that have been accepted, and in current use, since 1967. Before that time the technique was called isoelectric separation, isoelectric fractionation, isoelectric condensation, isoelectric analysis and focusing electrophoresis, as well as stationary electrolysis. Terms such as density gradient electrofocusing or gel electrofocusing indicate the medium in which the experiments are carried out. 

Figure 5. Two sucrose density gradient electrofocusing runs identical except for the polarity of the electrodes. The first, A, has been focused with the anode in the dense solution, i.e., at the bottom of the column, and the second, B, with the cathode in the dense solution. The pH-gradient is hardly affected by the superimposed sucrose density gradient. In the conductivity, however, there is a difference in the two experiments. With the cathode in the bottom, the conductivity lowering effect from the sucrose density gradient will add to the relatively low conductivity of the carrier ampholytes isoelectric near pH 9. In both curves one can observe the low conductivity of the carrier ampholytes around pH 7.

The stabilization techniques of importance today in electrofocusing are (a) density gradient electrofocusing, (b) gel electrofocusing, and (c) zone convection electrofocusing. These methods are described in detail later in this article. 

Figure 19. A density gradient electrofocusing experiment run in the apparatus depicted in Fig. 17 with the electric field on during elution according to Svendsen (128). The apparatus was fitted with a cold finger. The gradient had a height of 21 cm and a cross section of 7.2 cm. It was a run for 48 hours at 17°C. The protein load was 6.5 mg human hemoglobine cyanide. Elution was done with an input of 2.25 ml/h and an output of 13.7 ml/h. Fractions were taken so that one fraction corresponded to 0.1 cm column height. The fractions were read on a spectrophotometer. However, pH determination cannot be done since every sample is diluted with phosphoric acid during the elution. (Svendsen, 56).

If it is desired to make a closer study of a protein component after it has been electrofocused, a small piece of the gel can be cut out. The piece should be soaked in water and the extract should be measured. This technique has been employed by Awdeh and co-workers, Wrigley, and others. However, sucrose density gradient electrofocusing is probably preferable if more exact studies are necessary. Gel electrofocusing is cheaper and simpler. Thus it can serve as an excellent pilot for density electrofocusing, which is relatively expensive and time-consuming. 

Density gradient electrofocusing is very easily disturbed by large samples or large amounts of one component of a sample. Electrofocusing in polyacrylamide gel is not disturbed by laige samples or overloading. This has been demonstrated many times. 

Figure 35. Density gradient electrofocusing of part of defatted human serum albumin. The pH 5.2-S.9 gradient was prepared by the method of zone convection electrofocusing. (Valmet, 78.)...

Figure 34 shows a pH-gradient prepared from a fraction which originally had a range of 2 pH units. It was then used to analyze a sample by density gradient electrofocusing (see Figure 35). The sample consi.sted of serum albumin with pi values lying between pH 5.1 and 5.8 at 4 C (78).

Shackman JG, Ross D (2007) Counter-flow gradient electrofocusing. Electrophoresis 28(4) 556-571... 

The first practical IEF experiments were carried out with the use of synthetic molecules, called carrier ampholytes, to generate the pH gradients.1,26 Carrier ampholytes are amphoteric electrolytes that carry both current and buffering capacity. Much of the early theoretical activity in electrofocusing dealt with the properties required of carrier ampholytes and is more or less irrelevant to a current discussion.1,3,9 Different varieties of... 

E3. Ernes, E. V., Latner, A. L., and Martin, A. ]., Electrofocusing followed by gradient electrophoresis A two-dimensional polyacrylamide gel technique for the separation of proteins and its application to the immunoglobulins. Clin. Chim. Acta 64, 69-78 (1975). 

Sherbet and Lakshmi developed a small-size electrofocusing chamber (Moore and Hibbitt, 1975 Sherbet, 1978) made of Perspex (Figure 7). It consists of two vertical chambers and openings at the top that communicate at the bottom where a needle valve can close the entrance. The chamber of 1.4-cm i.d. contains the density gradient (2-1.5% Ficoll plus 1% ampholine), while the small-bore chamber (0.5-cm i.d.) contains an... 

Insertion of cells at low pH may result in the extraction of cellular proteins. For IEF of HeLa cell metaphase chromosomes, it was found that stripping of basic proteins occurred in the pH gradient (Landel et al., 1972). In this respect, Boltz et al. (1977) and Hammerstedt et al. (1979a) noted that the focusing pH value of sperm cells was related to the pH of sample injection, certainly due to the extraction of basic proteins at low pH. Also sperm became immotile after suspension in electrofocusing... 

For isolelectric focusing the sample is mixed into a linear sucrose gradient from 35 to 0% (w/v), containing 5% (by volume) of carrier ampholyte solution, pH 4—7, (equivalent to an ampholyte concentration of 2%, w/v) in a 110-ml electrofocusing column (LKB, Sweden). The cathode space is filled with 1.5% (w/v) of ethylene diamine in a 50% (w/v) sucrose solution the sample space is topped off with 0.1% aqueous H2SO4 as anode solution. Focusing is performed for 72 h at 500 V then the contents are collected in 3-ml fractions. 

Nguyen, N. Y., Salokangas, A., and Chrambach, A., 1977, Electrofocusing in natural pH gradients formed by buffets gradient modification, Anai. Biochem. 78 287-294. 

Figure 1. Three proteins, marked pli, pli, and pli, schematically electrofocused in a column. Each of the three protein components is negatively charged above its respective point and positively charged beneath. For that reason it will move in the electric field until the position in the column is reached where the pH of the pH-gradient is the same as the isoelectric point for that particular protein.

For the practical use of electrofocusing it must be possible to arrange a pH-gradient with suitable properties. The gradient should comprise the desired pH range and other parameters of importance as slope, and conductivity. The pH-gradient may be achieved in two different ways. 

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