Isoelectric focusing

Isoelectric focusing, IEF, (often called electrofocusing) is a method for separating molecules which differ in their charge characteristics. For IEF of proteins, the protein mixture is subjected to an electric field in an inert support in which a stable pH gradient has previously been generated. The anode region is at a lower pH than the cathode and the pH range is chosen such that the proteins to be separated have their isoelectric points within this range. A protein which is in a pH region below its pI will be positively charged and so will migrate towards the cathode. However, as it migrates, so the pH will decrease until the protein reaches a pH which is its pI. At this point it has no net charge and so migration ceases. Should it overshoot this point, it will enter a region of pH above its pI and so become negatively charged. It will then reverse its direction of migration and now migrate towards the anode. Therefore proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI. The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands.

The stable pH gradient between the electrodes is formed by including a mixture of low molecular weight 'carrier ampholytes' in the inert support. These are synthetic, aliphatic polyaminopolycarboxylic acids available commercially whose individual pI values cover a preselected pH range. Thus one can purchase carrier ampholytes spanning either a wide pH range (e.g. pH 3 - 10) or a narrow range (e.g. pH 7 - 8). Commercial ampholytes include Ampholine, BioLyte and Pharmalyte.

The exact format for IEF depends on the nature of the inert support which is used. Early applications of IEF used water-cooled vertical glass columns filled with carrier ampholytes in a sucrose density gradient. The sucrose density gradient was the inert support, the density gradient helping to stabilize the liquid column against convective mixing due to heat generated during electrophoresis. The upper electrode (anode) was connected to a reservoir of acid (e.g. phosphoric acid) and the lower electrode to a reservoir containing an alkaline solution (e.g. NaOH). After a period of electrophoresis to establish the pH gradient, the protein sample was introduced into the column and electrophoresis continued until the proteins had reached their pI's. The individual protein bands were then recovered by draining the column from its base into a series of fractions. This procedure is still used for some applications of preparative IEF although it is not widespread due to the high cost of the ampholytes used. Preparative IEF can also be carried out on a somewhat smaller scale (but still capable of producing milligram amounts of pure proteins) using horizontal flat beds of a gel bead matrix, such as Sephadex, in cooled glass troughs. After electrophoresis the gel is subdivided into fractions from which the proteins can be eluted with buffer.

Despite the above preparative approaches, IEF is mainly used as an analytical technique to assess the complexity or purity of protein samples. As with SDS-PAGE, however, if the amount of protein required in pure form is only of the order of a few micrograms, even analytical scale electrofocusing may be sufficient to prepare it. Analytical IEF is carried out either in vertical polyacrylamide rod gels (especially as the first-dimensional separation of two-dimensional gel electrophoresis) or in horizontal polyacrylamide or agarose slab gels.

Horizontal slab gel IEF is now the most widely used format for electrofocusing. Thin sheets of polyacrylamide or agarose gel mounted on glass or plastic plates and containing carrier ampholytes chosen to give the correct pH range can be purchased or prepared in the laboratory. It is important to avoid molecular sieving effects so that the protein separation occurs solely on the basis of charge. Hence agarose gels would be chosen in preference to polyacrylamide gels for the separation of larger proteins because of their larger pore size. If polyacrylamide gel is used, the gel concentration is chosen so as to give a large pore gel and so minimize molecular sieving. Gel thicknesses of 1 - 2 mm can be used but increasingly ultra-thin gels only 0.1 - 0.25 mm thick are being used since these are cheaper (carrier ampholytes are expensive) and allow shorter running times (since higher voltages can be used) and improved resolution.

The horizontal gels on their glass or plastic sheets are arranged on water-cooled plates since this allows the heat generated by electrophoresis to be readily dissipated and so avoid distortion of the separating protein bands. As with vertical polyacrylamide slab gels, multiple samples can be analysed side by side. Only a few micrograms of proteins are required for analysis. A simple procedure to load the samples onto the gel is to dip a small piece of filter paper into the sample and then lay this on the gel surface at the origin. A short period of electrophoresis now allows the proteins to enter the gel, the filter paper is removed and electrophoresis is continued for several hours to allow IEF to occur. The profile of separated proteins can be visualized after electrophoresis by fixing and staining the protein bands.

Unlike SDS-PAGE, IEF gives no information about the molecular weight of the separated polypeptides since separation occurs on the basis of charge alone. However, by measuring the pH gradient across the gel after electrophoresis using a surface electrode and recording the position of the separated protein of interest, one can estimate the pI of this protein; information which could be invaluable in planning any large-scale ion-exchange chromatography step in a purification scheme.

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