Preparative isoelectric focusing

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. 

EC 1.15.1.1]. Purified by DEAE-Sepharose and copper chelate affinity chromatography. The preparation was homogeneous by SDS-PAGE, analytical gel filtration chromatography and by isoelectric focusing [Weselake et al. Anal Biochem 155 193 1986 Fridovich J Biol Chem 244 6049 7969]. 

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. 

Figure 3. Preparative isoelectric focusing. The PNL eluted from gel filtration was subjected to isoelectric focusing using a column of 110 ml capacity (LKB) with ampholytes pH 7-11. After 48 h (9.6 W constant power), fractions of 3 ml were removed and assayed for PNL activity (- - ) and pH (- -).

However, after the preparative isoelectric focusing column, the PNL was the only band detected both by lyase activity staining (B 3) and by protein staining (a 3). 

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

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. 

Preparative electrophoresis on Sephadex G-25 (Ref. 168) or double isoelectric focusing,208 preceded by chromatography on Sephadex G-75, CM-cellulose, and calcium phosphate, was used for the isolation of endo-D-galacturonanase from the filtrate of a Verticillium albo-atrum culture. The homogeneity was confirmed in both cases by electrophoresis on poly(acrylamide) gel. The molecular weight of the enzyme was close to the values found for Aspergillus endo-D-galacturonanases. 

Electrophoretic Methods Systems for polyacrylamide gel electrophoresis, 104, 237 preparative isoelectric focusing,104, 256 gel... 

These two polypeptides have been shown to have identical amino acid composition (Table 9) but to differ from each other in sialic acid content. Da and D4 have 1 and 2 moles of sialic acid per mole of protein, respectively (A5, A6, B9, BIO, E5). A third form without sialic acid has been isolated by preparative isoelectric focusing (A5). Both D3 and D4 have the same NHa-terminal (serine) and COOH-terminal (alanine) amino acid residue and a molecular weight of about 10,000. The complete amino acid sequence has recently been announced (B6) and is reported in Fig. 7. These studies show that the polysaccharide having sialic acid as its terminal sugar, is linked to threonine 74 of the polypeptide chain. 

Although H. annosum has an extremely high variation in the isoenzyme pattern of other enzymes, it produced only a single band in the isoelectric focusing experiment of the laccase preparations isolated from more than 60 different strains (20). 

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