Separations isoelectric focusing

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,... 

D SDS-PAGE which combines tw o modes of separation isoelectric focusing and polyacrylamide gel elctrophoresis has been on the market for about 30 years (OTarrell, 1975 Barritaultetal., 1976). Initially this technique didnot gain much of popularity due to obstacles in part originating from poor... 

Figure 1. Sepaiatioii of protein by one and two imensional electrophoresis. A sample of mouse urine was separated by SDS-PAGE (size-based separation), isoelectric focusing (charge based separation) and by a two dimensional (charge, then size) separation. Proteins were visualised by staining with Coomassie blue protein stain or silver.

There are three distinct modes of electrophoresis zone electrophoresis, isoelectric focusing, and isotachophoresis. These three methods may be used alone or in combination to separate molecules on both an analytical (p.L of a mixture separated) and preparative (mL of a mixture separated) scale. Separations in these three modes are based on different physical properties of the molecules in the mixture, making at least three different analyses possible on the same mixture. 

Isoelectric Focusing. Isoelectric focusing is a technique used for protein separation, by driving proteins to a pH where they have no mobiUty. Resolution depends on the slope of a pH gradient that can be achieved in a gel. 

In considering the applicability of preparative classical electrophoretic methods to chiral separations, it should be noted that practitioners in the art of classical electrophoresis have been particularly inventive in designing novel separation strategies. For instance, pH, ionic strength and density gradients have all been used. Isoelectric focusing and isotachophoresis are well-established separation modes in classical electrophoresis and are also being implemented in CE separations [7, 8]. These trends are also reflected in the preparative electrophoretic approaches discussed here. 

Righetti and co-workers [11] were one of the first to demonstrate the utility of classical isoelectric focusing for the chiral separation of small molecules in a slab gel configuration. In their system, dansylated amino acids were resolved enan-tiomerically through complexation with (i-cyclodextrin. Preferential complexation between the cyclodextrin and the derivatized amino acid induced as much as a 0.1 pH unit difference in the pK s of the dansyl group. 

Two-dimensional gel electrophoresis (2DE) is a two-dimensional technique for protein separation, which combines isoelectric focusing and sodium dodecyl sulphate (SDS) electrophoresis. The high resolving power results from separation according to charge (isoelectric point) in the first dimension and size (mobility in a porous gel) in the second dimension. Depending on the gel size, from several hundred to more than 5,000 proteins can be separated. 

Rossieretal. [332] usedUV excimer laser photoablation to cut channels 50 microns deep by 100 microns wide in laminated PET. These channels were filled with PA, and rapid separation of proteins by isoelectric focusing was demonstrated. 

Separation of the purified PL1, PL2 and PL3 isoenzymes by SDS-PAGE yielded single protein bands that corresponded to a molecular mass of 42 kDa (6), 4 kDa higher than calculated from the deduced amino acid sequences. Isoelectric focusing revealed a pi of >10 for each isoenzyme similar to that of the basic Ech PLs (15). 

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

Three bacterial species (E. coli, P. putida, and S. rubidae) were separated on isoelectric focusing in methylcellulose coated capillaries, and three bacterial species (P. fluorescens, E. aerogenes, and M. luteus) and the yeast S. cerevisae, were separated by capillary electrophoresis in the presence of polyethylene oxide.101 The polymers served to minimize adsorption to the walls without causing cellular lysis. 

Glukhovskiy P. and Vigh G., Analytical- and preparative-scale isoelectric focusing separation of enantiomers, Anal. Chem. 71, 3814, 1999. 

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