Isoelectric focusing considerations

Most electrophoretic methods have been tried in a free-flow format, including isoelectric focusing, native zone electrophoresis, and isotachophoresis. Most free-flow electrophoresis equipment has very low (ca 1 g/(L-h)) capacity, and resolution is reduced by heating and electroosmotic considerations. 

Manning JM et aJ Normal and abnormal protein subunit interactions in hemoglobins.] Biol Chem 1998 273 19359-Mario N, Baudin B, Giboudeau J Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing. J Chromatogr B Biomed Sci Appl 1998 706 123-Reed W, Vichinsky EP New considerations in the treatment of sickle cell disease. Annu Rev Med 1998 49 46l. 

Most previous work on rat MUPs has been conducted with inbred or relatively inbred laboratory rat strains that are likely to exhibit considerably reduced phenotypic variation relative to the wild population, as we see in mice. As an initial exploration of MUP expression, we analysed urine from nine wild-caught male rats captured from several different populations in northern UK by isoelectric focusing electrophoresis (IEF). The protein banding pattern was very similar between individuals, consisting of two major and several minor bands. Peptide mass... 

Zone electrophoresis is influenced by adsorption and capillarity, as well as by electroosmosis. Therefore evaluation of mobility (and f) from this type of measurement is considerably more complex than from either microelectrophoresis or moving-boundary electrophoresis. Nevertheless, zone electrophoresis is an important technique that is widely used in biochemistry and clinical chemistry. One particularly important area of application is the field of immunoelectrophoresis, which is described briefly in Section 12.11. Additional information on zone electrophoresis may be obtained from Probstein (1994) and Hunter (1981) and the references given there. Variants of zone electrophoresis also exist see, for example, Gordon et al. (1988) for information on a variant known as capillary zone electrophoresis and Righetti (1983) for information on what is known as isoelectric focusing. 

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. 

Capillary electrophoretic methods including open-column zone electrophoresis, disc electrophoresis in gels, isotachophoresis and isoelectric focusing have received considerable attention from the analytical community over the last three or four years (80, 81, 82). In capillary zone electrophoresis (CZE), nanogram quantities of sample are placed in a silica capillary, 50 to 300 miaons in diameter and 50 to 100 cm long. Since the small dimensions of the capillary allow for efficient removal of Joule heat, electrical fields up to 350 V/cm can be applied. Under the influence of the field, sample components separate by zone electrophoresis while they are carried downstream by electro-osmosis. 

The terms cellulolytic enzymes and cellulases now enjoy a workable definition. This has been made possible through the separation of the components of the cellulolytic complex, as well as some clarification of their mechanism of action. New viscometric, isoelectric focusing, gel filtration, and other techniques for the determination of the enzymic components have been devised. Detailed observations on the molecular sites of hydrolytic reactions, the susceptibility of the enzymic reactions to metallic ions, proteolytic enzymes, and other chemicals also provided considerable insight into the nature of cellulase. 

Poly(A)-rich mRNA prepared from rabbit pancreas directed the synthesis of a protein (mol. wt. 5.65 X 10 ), which, using a cell-free protein synthesizing system derived from wheat embryo, appeared to represent the product of o-amylase mRNA cell-free translation.This protein, which was 1500 daltons larger than the purified rabbit pancreas a-amylase, was resolved by isoelectric focusing into discrete species similar to those observed for purified a-amylase. The protein, which on peptide analysis demonstrated considerable identity with the a-amylase, was selectively precipitated by glycogen, but with a much lower recovery than that observed for rabbit on porcine pancreatic a-amylases. 

In a study of the secretion of lysosomal hydrolases by stimulated and non-stimulated macrophages from mice, a-D-mannosidase was found to be secreted in a considerable amount by a mechanism independent of phagocytosis. a-D-Mannosidase solubilized by 3M-LiCl from the cell walls of the coleop-tiles of Avena sativa has been purified via fractional precipitation, gel filtration, ion-exchange chromatography, and isoelectric focusing to homogeneity on disc gel electrophoresis. The enzyme (mol. wt. 6.3 x 10, pH optimum 4.5, 3.2 mM for 4-nitrophenyl a-D-mannopyranoside) had no metal ion... 

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