The Principles of Isoelectric Focusing

The net charge on a protein is the algebraic sum of all its positive and negative charges. There is a specific pH for every protein at which the net charge it carries is zero. This isoelectric pH value, termed the isoelectric point, or pi, is a characteristic physicochemical property of every protein. The definition of pi for molecules as complex as proteins is more or less an operational one and is taken to be that pH at which a protein has zero electrophoretic mobility in an isoelectric focusing run. Nevertheless, it has been shown that the pis of some acidic proteins (up to about pH 7) can be calculated from their amino acid compositions.3 5 

Isoelectric focusing was developed to separate proteins on the basis of differences in their pi values. The mechanism of IEF can be treated mathematically and the classical analysis is presented in the Appendix. On the other hand, the mechanism of IEF is relatively easy to conceptualize and lends itself to a simple description. The historical, theoretical, and practical aspects of IEF are well documented. Many experimental details can be found in several monographs and review articles.1,2,8-17 Refs. 1 and 2 are particularly recommended. They provide valuable insights from one of the pioneers in the field. 

IEF is a high-resolution technique generally carried out under nondenaturing conditions, in which proteins maintain most of their physical and chemical 

Preparative electrofocusing is now most commonly done in free solution in specially designed chambers made up of interconnected cells that hold the focused, separated protein zones until they are collected.18-21 The separation axes of the most popular preparative cells are horizontal to minimize the effects of gravity on focused bands. 

Very often, electrofocused proteins show patterns of multiple bands in places where only a single band is expected. This phenomenon is called microheterogeneity. Early discussions of microheterogeneity attempted to explain it in terms of denaturation or of variable interactions between carrier 

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Figure 4.11. The Principle of Isoelectric Focusing. A pH gradient is established in a gel before loading the sample. (A) The sample is loaded and voltage is applied. The proteins will migrate to their isoelectric pH, the location at which they have no net charge. (B) The proteins form bands that can be excised and used for further experimentation.

The principle of this method is based on the fact that amphoteric macromolecules exhibit zero mobility in the electric field at their isoelectric points and tend to focus into narrow zones in places where the pH of the surrounding media equals p7. The process of isoelectric focusing involves two steps, namely the formation of a stable pH gradient that increases from the anode to cathode, and electrophoretic migration of the amphoteric molecules towards their pi positions with subsequent attainment... 

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. 

Fig. 4. Principle of isoelectric focusing. The sample is applied to a separation column in a mixture of carrier ampholytes. When a current is applied, the ampholytes form a continuous, stable and linear pH gradient, and the charged sample components move towards the electrode of the opposite charge. The individual components gradually lose their charge during migration until the charge is zero and movement stops at the pH corresponding to the isoelectric point (pi). S is the sample to be separated. A, B denote sample constituents after separation in the position of their pi.

Isoelectric focusing (IEF) is a steady-state zonal electrophoretic technique. Using IEF, proteins can be separated according to differences in their isoelectric points. The principles of IEF are described in Sections 8.10 and 8.11. 

The next four chapters discuss the basic principles underlying operation and method development of the most common electrodriven analytical techniques CE, capillary isoelectric focusing (cIEF), capillary gel electrophoresis (CGE), and affinity capillary electrophoresis (ACE). Weinberger presents a comprehensive approach for method development in CE with an emphasis on small-molecule applications. This is followed by Kilar s chapter describing the principles of and method development in cIEF, as well as recent innovations... 

A second example of the improvements in isoelectric focusing generated by the introduction of IPG gels is shown in Figure 11.8, where protein samples obtained from bean seeds are subjected to IEF for up to 12 h, using carrier ampholytes and immobilized pH gradients.11 In principle, proteins focused at their isoelectric pH values are not subject to changes in position with time, since IEF is a steady... 

The most powerful two-dimensional systems are those employing combinations of isoelectric focusing and various forms of polyacrylamide gel electrophoresis (PAGE) and these are described in the following sections. In particular, this section of the review will be concerned with the principles and applications of lEF/PAGE. 

Isoelectric Focusing. The principle of the isoelectric focusing method is to focus proteins at their respective pis (isoelectric points) in a stable... 

In microfluidic-based systems, material is transported within microstructures (of typical dimensions of 10-500 pm) where separations, reactions, and other processes occur. Focus has been on the realization of the traditional separation techniques (electrophoresis, chromatography, isoelectric focusing, etc.) and reactions in the microchip format. The principles of separation, as in the conventional formats, are based on differences in mass and charge (thus mobility) and partitioning between phases. However, advantages associated with the small dimensions provide superior performance. For example, the higher surface to volume ratio arising from the smaller dimensions results in lower heat and mass transfer resistances and thus an improved performance. 

Svensson (1961) and subsequently Vesterberg (1969) developed the important technique of isoelectric focusing (lEF). This method exploits the principles of moving boundary electrophoresis. Components are separated according to their pi by the use of carrier ampholytes in the supporting medium. An ampholyte is a compound that can have basic and acidic properties, such as amino acids. The migration of ampholytes is therefore pH dependent. This method has been combined with gel electrophoresis to form the powerful tool of two-dimensional gel electrophoresis pioneered by O Farrell in 1975. 

Capillary isoelectric focusing separates analytes based on differences in their isoelectric points (pi) using the same principles as in preparative solution IEF. After a focusing step, that builds up a linear pH gradient in the capillary (controlled with zwitterionic internal markers), the analytes move as a function of their respective charge until they reach a position of zero charge (isoelectric point). The solution is then mobilized in CIEF to the detector hydrodynamically. 

CE has many separation modes that are beneficial to protein impurity analysis. Within the many thousands of potential protein impurities in a recombinant product there will be several that have only minor physicochemical differences from the drug product. The application of different CE modes can potentially resolve these impurities. CE methods can be divided into four principle modes that are applicable to recombinant protein impurity analysis capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, and micellar electrokinetic capillary chromatography. Each mode will be discussed briefly. Since the technology is so young and still very exploratory, CE methods are developed empirically for specific separations. It is difficult to provide standard protocols for CE impurity analysis. Instead, protocols that can be used as a starting point for impurity analysis will be provided as well as the citation of examples of impurity analyses from the literature to provide additional sources of protocols for interested readers. 

The most distinguishing phenomenon in capillary electrophoresis is whether the experiments are performed in the absence or in the presence of electroos-motic flow (EOF), (see Chapter 6 for details on EOF). Unlike other types of capillary electrophoresis, isoelectric focusing can be performed under both modes. Since the experimental and theoretical principles governing these modes of CIEF are different, they will be discussed separately. 

The pH of a slurry has a profound influence on its colloidal stability and CMP performance. Strong correlations have been established between the particle isoelectric point (lEP) and the optimal pH for slurry stability. The general rule is that the slurry is more stable at a pH that is away from the lEP, so the zeta potential of the particles is greater than 20 mV. The focus of this section is on the influence of pH on the slurry performances such as material removal rate and defectivity. In order to examine the impact of slurry pH on these two important performance features, we first take a closer look at the interaction between abrasive particles and the surface to be polished. There is a vast amount of literature on the interaction between abrasive particles and silicon dioxide surface [26]. The discussion below will focus on the interaction between ceria abrasive particles and the silicon dioxide surface to be polished. The basic principles and conclusions can be easily extended to other pairs of abrasive particles and surfaces. 

Electrokinetics play Important roles in colloid and surface science but also beyond these domains. The purpose of the present section is to consider some principles behind their measurements, emphasizing methods relevant for characterizing colloids and interfaces. The large number of semlquantitative methods, including paper or gel electrophoresis for diagnostic purposes and isoelectric focusing in protein chemistry, are considered "applications (sec. 4.10). For more details, see the literature of sec. 4.11, especially the books by Hunter, and Rlghettl et al. Unless where explicitly mentioned otherwise, the considerations apply to aqueous systems. 

One of the most effective methods. In terms of sample capacity, for large scale Isoelectric focusing utilizes membranes to define subcompartments In an electrolyzer. The membranes prevent bulk flow between adjacent compartments while allowing the free migration of proteins. Rllbe has described several devices based on this principle (20). The most recent Is a 7.6 liter cell with 46 separation compartments (21). It has a cylindrical geometry with closed compartments. The contents of each compartment are effectively mixed and cooled by the slow rotation of the submerged apparatus In a tank of cold water. The device has fractionated 14g of whey protein Into the major components, albumin (pi 4.6), alpha-lactalbumln (pi =... 

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