Fractionation methods isoelectric focusing

Electrophoretic Methods. Little use has been made of electrophoretic techniques for the fractionation of the whey proteins. Column isoelectric focusing has been used to fractionate further the crude immunoglobulin fraction obtained by Smith s procedure (Josephson et al. 1972). Two major peaks, a shoulder, and two minor peaks were obtained, but no attempt was made to identify the components in the peaks. 

The development of electrophoretic techniques afforded possibilities for fractionations based on charge density differences. Duxbury (1989) has reviewed applications of different electrophoretic separation methods, including zone electrophoresis, moving boundary electrophoresis, isotachophoresis, and isoelectric focusing (IEF). Preparative column electrophoresis (Clapp, 1957) and continuous flow paper electrophoresis (Hayes, 1960 summarized by Hayes et al., 1985) methods have been used to separate components isolated from sapric histosol soils. These techniques allowed separation of polysaccharides from the colored components the electrophoretograms of the colored components were diffuse, showing a continuum of components of different charge densities. 

In some steady-state methods of separation (isoelectric focusing, density-gradient centrifugation, and sometimes elutriation), component zones approach a stationary configuration centered about different points in space. Separation occurs by virtue of the different steady-state positions of the various solutes. In other systems (field-flow fractionation, zone refining,... 

Isoelectric focusing is a mature separation technique that has a place in any laboratory doing work with proteins. The analysis of a protein is not complete without a determination of its isoelectric point and all protein databases have at least estimates of the pis of the represented proteins. Proteins thought to be pure by other methods are often found to be mixtures of several proteins when analyzed by IEF. Isomeric forms of the same protein that are revealed by IEF are valuable indicators of mutations or differences in posttranslational modifications. IEF plays a crucial role in 2-D PAGE and preparative IEF allows for high-purity fractionations of unparalleled resolution. A simple keyword search of literature databases shows that about 500 journal articles are written per year referring to IEF. This amply attests to the value of IEF as a tool for protein analysis and purification. 

H. Haglund, Isoelectric Focusing in pH Gradients— A Technique for Fractionation and Characterization of Ampholytes, in Methods of Biochemical Analysis, Vol. 19 (D. Glick, Ed.), Wiley, New York, 1971, p. 1. 

This chapter will describe in detail the procedure for Western blotting of polypeptides and proteins separated on a denaturing polyacrylamide gel system. This procedure is used routinely in our laboratory for the analysis of polypeptides from a variety of subcellular fractions of whole tissue and cell lines, and has evolved over a number of years in the hands of several people. Many different immunoblotting procedures are currendy available details of variadons from the I-protein-A method described here are given in the Notes secdon, as are brief amendments covering electrotransfer from two-dimensional and isoelectric focusing systems. A detailed descripdon of sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) is not appropriate for this chapter, and the reader is referred to vol. 1, Chapter 6, and refs. 9-14. For details of the producdon of polyclonal and monoclonal andsera, Chapters 1-6 in this vol. 

Since this review was completed, a number of interesting developments have been obtained in this laboratory and elsewhere in various protein fractionation techniques at subzero temperatures. Let us mention a work on isoelectric focusing and electrophoresis by M. Per-rella, A. Heyda, A. Mosca and L. Rossi-Bemardi (Anal. Biochem., in press) a work by C. Le Peuch and C. Balny on the sequential elution of proteins bound by hydrophobic interaction chromatography [FEBS Lett. 87,232(1978)] a work by K. Andersson, Y. Benyamin, P. Douzou, and C. Balny about organic solvents and temperature effects on desorption from immunoadsorbents (J. Immunol. Methods, in press, 1978). 

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... 

Isoelectric focusing (IEF) is unique among separation processes as it results in a stationary steady state distribution of fractions along the column axis. The final distribution of fractions is independent of their initial distribution. As such, IEF has no analogue in other electrophoretic or chromatographic methods and well deserves its current popularity (2). 

The most widely used methods by far employ coherent or granular gels. The former are most commonly polyacrylamide or agarose and the latter is usually Sephadex. Gels have been used in the preparative applications of each of the three common electrophoretic modes, isoelectric focusing (lEF), isotachophoresis (ITP) and zone electrophoresis (ZE). Regardless of the mode utilized, the procedure is most often a batch operation and employs either a cylindrical flat bed or annular separation chamber. Isotachophoresis has been applied to preparative scale fractionations in both polyacrylamide (.3) and Sephadex... 

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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