Isoelectric focusing principles

Ek, K., Bjellqvist, B., Righetti, R G. (1983). preparative isoelectric-focusing in immobilized pH Gradients. 1. General principles and methodology. J. Biochem. Bioph. Meth. 8(2), 135-155. 

This introduction was intended to merely provide basic concepts and principles of electrophoretic separation. It should be noted that a wide variety of electrophoretic techniques, such as isoelectric focusing and two-dimensional gel... 

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. 

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. 

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. 

Laas, T. (1989). Isoelectric focusing in gels. In Protein Purification Principles, High Resolution Methods, and Applications (J.-C. Janson and L. Ryden, eds.), pp. 376-403. VCH Press, Weinheim. 

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 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... 

Westeemeiee, R., Postel,W. (1982). Isoelectric focusing in immobilized pH gradients principle, methodology and some applications. /. Biochem. Biophys. Methods 6(4), 317-339. 

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. 

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.

Polar fishes, freezing resistance of, 195 12-Propanediol-water mixture dielectric constant of, 92 Protein fractionation at subzero temperatures, 77 -189 acid-base equilibria, 100-122 applications, 146-185 column chromatography, 140-141 density and viscosity changes, 82-85 dielectric constant variations, 85-99 general principles, 135-140 isoelectric focusing, 141-144 methods, 140-146 physicochemical data, 78-134 protein dissociation, 129-134 protein titration, 116-122 solubility of salts and solutes, 122-129... 

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. 

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 =... 

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

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... 

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