This method is ideal for the separation of amphoteric substances (which behave as acids and as bases) such as proteins because it is based on the separation of molecules according to their different isoelectric points (pIs). The method has high resolution, being able to separate proteins that differ in their isoelectric points by as little as 0.01 of a pH unit. The most widely used system for isoelectric focussing (IEF) utilises horizontal gels on glass plates or plastic sheets. Separation is achieved by applying a potential difference across a gel that contains a pH gradient. The pH gradient is formed by the introduction into the gel of compounds known as ampholytes, which are complex mixtures of synthetic polyamino-polycarboxylic acids (Figure 1). Ampholytes can be purchased in different pH ranges covering either a wide band (e.g. pH 3–10) or various narrow bands (e.g. pH 7–8), and a pH range is chosen such that the samples being separated will have their isoelectric points within this range. Commercially available ampholytes include BioLyte ® and Pharmalyte ®.

Fig1. The general formula for ampholytes.

Traditionally, 1–2 mm thick IEF gels have been used by research workers, but the relatively high cost of ampholytes makes this a fairly expensive procedure if a number of gels are to be run. However, the introduction of thin-layer IEF gels, which are only 0.15 mm thick and which are prepared using a layer of electrical insulation tape as the spacer between the gel plates, has considerably reduced the cost of preparing IEF gels, and such gels are now commonly used. Since this method requires the proteins to move freely according to their charge under the electric field, IEF is carried out in low-percentage gels to avoid any sieving effect within the gel. Polyacrylamide gels (4%) are commonly used, but agarose is also used, especially for the study of high M r proteins that may undergo some sieving, even in a low-percentage acrylamide gel.

To prepare a thin-layer IEF gel, carrier ampholytes, covering a suitable pH range, and riboflavin are mixed with the acrylamide solution, and the mixture is then poured over a glass plate (typically 25 cm × 10 cm), which contains the spacer. The second glass plate is then placed on top of the first to form the gel cassette, and the gel photopolymerised by placing it in front of a bright light. The photodecomposition of the riboflavin generates a free radical, which initiates polymerisation; this takes 2–3 h. Once the gel has set, the glass plates are prised apart to reveal the gel stuck to one of the glass sheets. Electrode wicks, which are thick (3 mm) strips of wetted filter paper (the anode chamber contains phosphoric acid, the cathode chamber sodium hydroxide) are laid along the long length of each side of the gel and a potential difference applied. Under the effect of this potential difference, the ampholytes form a pH gradient between the anode and cathode. The power is then turned off and samples applied by laying on the gel small squares of filter paper soaked in the sample. A voltage is again applied for about 30 min to allow the sample to electrophorese off the paper and into the gel, at which time the paper squares can be removed. Depending on which point on the pH gradient the sample has been loaded, proteins that are initially at a pH region below their isoelectric point will be positively charged and will initially migrate towards the cathode. As they proceed, however, the surrounding pH will be steadily increasing, and therefore the positive charge on the protein will decrease correspondingly until eventually the protein arrives at a point where the pH is equal to its isoelectric point. The protein will now be in the zwitterion form with no net charge, so further movement will cease. Likewise, substances that are initially at pH regions above their isoelectric points will be negatively charged and will migrate towards the anode until they reach their isoelectric points and become stationary. It can be seen that as the samples will always move towards their isoelectric points it is not critical where on the gel they are applied. To achieve rapid separations (2–3 h) relatively high voltages (up to 2500 V) are used. As considerable heat is produced, gels are run on cooling plates (10 °C) and power packs used to stabilise the power output to minimise thermal fluctuations. Following electrophoresis, the gel must be stained to detect the proteins. However, this cannot be done directly, because the ampholytes will stain too, giving a totally blue gel. The gel is therefore first washed with fixing solution (e.g. 10% (v/v) trichloroacetic acid). This precipitates the proteins in the gel and allows the much smaller ampholytes to be washed out. The gel is stained with Coomassie Brilliant Blue and then destained. A typical IEF gel is shown in Figure2.

Fig2. A typical isoelectric focussing gel. Track 1 contains a mixture of standard proteins of known isoelectric points. Tracks 2–5 show increasing loadings of venom from the Japanese water moccasin snake. (Courtesy of Bio-Rad Laboratories Ltd.)

The pI of a particular protein may be determined conveniently by running a mixture of proteins of known isoelectric points on the same gel. A number of mixtures of proteins with differing pI values are commercially available, covering the pH range 3.5–10. After staining, the distance of each band from one electrode is measured and a graph of distance for each protein against its pI (effectively the pH at that point) plotted. By means of this calibration line, the pI of an unknown protein can be determined from its position on the gel.

IEF is a highly sensitive analytical technique and is particularly useful for studying microheterogeneity in a protein. For example, a protein may show a single band on an SDS gel, but may show three bands on an IEF gel. This may occur, for example, when a protein exists in mono-, di- and tri-phosphorylated forms. The difference of a couple of phosphate groups has no significant effect on the overall relative molecular mass of the protein, hence a single band on SDS gels, but the small charge difference introduced on each molecule can be detected by IEF.

The method is particularly useful for separating isoenzymes, which are different forms of the same enzyme, often differing by only one or two amino-acid residues. Since the proteins are in their native forms, enzymes can be detected in the gel either by washing the unfixed and unstained gel in an appropriate substrate or by overlayering with agarose containing the substrate. The approach has found particular use in forensic science, where traces of blood or other biological fluids can be analysed and compared according to the composition of certain isoenzymes.

Although IEF is used mainly for analytical separations, it can also be used for preparative purposes. In vertical column IEF, a water-cooled vertical glass column is used, filled with a mixture of ampholytes dissolved in a sucrose solution containing a density gradient to prevent diffusion. When the separation is complete, the current is switched off and the sample components run out through a valve in the base of the column. Alternatively, preparative IEF can be carried out in beds of granulated gel, such as Sephadex ® G-75.

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Androgen Biosynthesis

Androgens and Related Molecules

Two-Dimensional Polyacrylamide Gel Electrophoresis

Native (Buffer) Gels

Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis

Transition Metals are Essential for Health

Individual Regulatory Events Combine to Form Sophisticated Control Networks

Regulation of Enzyme Quantity

Biochemical Evaluation of Cobalamin and Folate Deficiencies

Compartmentation Ensures Metabolic Efficiency & Simplifies Regulation

Regulation of Metabolite Flow can be Active or Passive

Catabolism of Catecholamines