Introduction

Isoelectric focusing (IEF) is an electrophoretic method that separates proteins according to their isoelectric points (pI). Proteins are amphoteric molecules; they carry either positive, negative, or zero net charge, depend-ing on their amino acid composition and the pH of their surroundings. The net charge of a protein is the sum of all the negative and positive charges of its amino acid side chains and amino- and carboxyl-termini.

The isoelectric point is the specific pH at which the net charge of the protein is zero. Proteins are positively charged at pH values below their pI and negatively charged at pH values above their pI (Fig 3.1).

The presence of a pH gradient is critical to the IEF technique. In a pH gradient, under the influence of an electric field a protein will move to the position in the gradient where its net charge is zero. A protein with a positive net charge will migrate toward the cathode, becoming progressively less positively charged as it moves through the pH gradient until it reaches its pI. A protein with a negative net charge will migrate toward the anode, becoming less negatively charged until it also reaches zero net charge. If a protein should diffuse away from its pI, it immediately gains charge and migrates back to its isoelectric position. This is the focusing effect of IEF, which concentrates proteins at their pIs and separates proteins with very small charge differences. Because the degree of resolution is determined by electric field strength, IEF is performed at high volt-ages (typically in excess of 1 000 V). When the proteins have reached their final positions in the pH gradient, there is very little ionic movement in the system, resulting in a very low final current (typically below 1 mA).

One method for generating pH gradients in IEF gels relies on carrier ampholytes. Carrier ampholytes are small, soluble, amphoteric molecules with a high buffering capacity near their pI. Commercial carrier ampholyte mixtures comprise hundreds of individual polymeric species with pIs spanning a specific pH range. When a voltage is applied across a carrier ampholyte mixture (Fig 3.2) , the carrier ampholytes with the lowest pI (and the most negative charge) move toward the anode, and the carrier ampholytes with the highest pI (and the most positive charge) move toward the cathode. The other carrier ampholytes align themselves between the extremes, according to their pIs, and buffer their environment to the corresponding pH. The result is a continuous pH gradient.

IEF can be run in either a native or a denaturing mode. Native IEF is the more convenient option, as precast native IEF gels are available in a variety of pH gradient ranges. This method is also preferred when native protein is required, as when activity staining is to be employed. The use of native IEF, however, is often limited by the fact that many proteins are not soluble at low ionic strength or have low solubility close to their isoelectric point. In these cases, denaturing IEF is employed. Urea is the denaturant of choice, as this uncharged compound can solubilize many proteins not otherwise soluble under IEF conditions. Detergents and reducing agents are often used in conjunction with urea for more-complete unfolding and solubilization. Urea is not stable in aqueous solution, so precast IEF gels are not manufactured with urea. Dried precast gels are a convenient alternative; they have been cast, rinsed, and dried and can be rehydrated with urea, carrier ampholytes, and other additives before use.

Fig 3.1. Net charge on a protein as a function of pH. In this example the protein has a net charge of +2 at pH 5.5, 0 at pH 7.5 (the isoelectric point), and -1 at pH 8.5.	Fig 3.2. Creating a carrier ampholyte pH gradient. (A) No voltage applied. (B) Ampholytes and proteins move by electrophoresis when charged. (C) At isoelectric pH, ampholytes and proteins are focused.

Although useful, carrier ampholytes have some limitations. Because the carrier ampholyte–generated gradient is dependent on the electric field, it breaks down when the field is removed. The pH gradients are also susceptible to gradient drift (or cathodic drift), a phenomenon in which there is a gradual decrease in pH at the cathodic (–) end of the gel and a flattening out of the pH at the anodic (+) end. For this reason it is important to not overfocus the protein, because cathodic drift will increase over time. There can be significant batch-to-batch and company-to-company variations in the properties of carrier ampholytes, which limits the reproducibility of focusing experiments. Another problem encountered with carrier ampholytes is their tendency to bind to the sample proteins, which may alter the migration of the protein and render the separation of carrier ampholytes from the focused protein difficult.

Acrylamido buffers are an alternative means to form pH gradients that circumvent most of the limitations of carrier ampholytes. Chemically, they are acrylamide derivatives of simple buffers and do not exhibit amphoteric behavior. The acrylic function of an acrylamido buffer co-polymerizes with the gel matrix and, by pouring a gel that incorporates an appropriate gradient of acrylamido buffers, an immobilized pH gradient (IPG) is formed (Fig 3.3). The protein sample can be applied immediately (no prefocusing is needed). The pH gradient is stable and does not drift in an electric field. Additionally, the gels are not susceptible to cathodic drift, because the buffers that form the pH gradient are immobilized within the gel matrix. Acrylamido buffers are available from Amersham Pharmacia Biotech as individual Immobiline ™ species with a specific pK value (or optimum pH buffering range), suitable for casting gradients from pH 3–10.

Because reproducible linear gradients with a slope as low as 0.01 pH units/cm can separate proteins with pI differences of 0.001 pH units, the resolution possible with immobilized pH gradient gels is 10–100 times greater than that obtained with carrier ampholyte–based IEF. IEF is best performed in a flatbed electrophoresis apparatus. This type of apparatus allows very effective cooling, which is necessary due to the high voltages employed for IEF. Amersham Pharmacia Biotech offers a variety of precast gels for IEF, including ready to use carrier ampholyte gels, dried IPG gels, and dried acrylamide gels ready for reswelling in a mixture of carrier ampholytes and any other additives desired, such as detergent and denaturants.

Fig 3.3. Creating an immobilized pH gradient. (A, B, C) A gradient of acrylamido buffers in an acrylamide solution is cast into a slab gel that is crosslinked to a plastic support film. (D) The gel is washed to remove polymerization byproducts. (E) The gel is dried for storage. (F) The pH at any point in the gel is determined by the mixture of buffers crosslinked into the gel at that site.

Table 3.1 lists the pH ranges available for these gels.

Table 3.1 . Precast IEF gels for Multiphor™ II Flatbed Electrophoresis System Gel type

Ampholine™ PAGplate IEF pH 3.5–9.5 pH 4.0–6.5 pH 5.5–8.5 pH 4.0–5.0	CleanGel™ Dry IEF
For all gels: sample volume, 1–40 µl; number of samples/gel, 26–52; samples are applied either by application strips or pieces.