Isoelectric focusing

The principle behind lEF separations is described in Section 6.3. The separation is according to the isoelectric points. Isoelectric focusing in CE is used for protein 

1) Sample application A mixture of sample and ampholyte solution is filled into the 

2) Focusing High voltage is applied, and the ampholytes form a pH gradient in the capillary. The sample components move and are focused at their respective pi point. In the focusing step, the anode is placed in a low-pH electrolyte solution, while the cathode is placed in a high-pH electrolyte solution. 

3) Mobilization (for detection) The cathode electrolyte solution is changed to one with lower pH, and the analytes will migrate toward the cathode (and pass the detector) due to changes in the pH gradient. 

Electroosmotic flow is unwanted in lEF, since the EOF will disturb the focusing. Hence, surface-modified capillaries are used, that is, capillaries where the surface has been modified to carry no charge, to eliminate EOF, and also to avoid adsorption 

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 

At or in the proximity of their pi values proteins exhibit a minimum total charge and reduced solubility in the electrolyte solution [350,370,385]. This increases the probability of aggregation, and is further enhanced by the low ionic strength of the ampholyte buffer. Under these conditions protein precipitation results from hydrophobic interactions. These interactions can be suppressed by addition of additives such as ethylene glycol (10-40 %), non-ionic or zwitterionic surfactants (1-4 %), or sorbitol to the ampholyte buffer. 

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Bacteria produce chromosomady and R-plasmid (resistance factor) mediated P-lactamases. The plasmid-mediated enzymes can cross interspecific and intergeneric boundaries. This transfer of resistance via plasmid transfer between strains and even species has enhanced the problems of P-lactam antibiotic resistance. Many species previously controded by P-lactam antibiotics are now resistant. The chromosomal P-lactamases are species specific, but can be broadly classified by substrate profile, sensitivity to inhibitors, analytical isoelectric focusing, immunological studies, and molecular weight deterrnination. Individual enzymes may inactivate primarily penicillins, cephalosporins, or both, and the substrate specificity predeterrnines the antibiotic resistance of the producing strain. Some P-lactamases are produced only in the presence of the P-lactam antibiotic (inducible) and others are produced continuously (constitutive). 

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. 

Isoelectric focusing takes along (from ca 3 to 30 h) time to complete because sample compounds move more and more slowly as they approach the pH in the gel that corresponds to their isoelectric points. Because the gradient ampholytes and the samples stop where they have no mobiHty, the resistivity of the system increases dramatically toward the end of the experiment, and the current decreases dramatically. For this reason, isoelectric focusing is usually mn with constant voltage. Constant current appHcation can lead to overheating of the system. 

Another form of ief is a method called direct tissue isoelectric focusing (dtif) (29) where isoelectric focusing in agarose is used to evaluate tissues. 

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. 

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]. 

Metallothionein (from rabbit liver) [9038-94-2], Purified by precipitation to give Zn- and Cd-containing protein fractions and running on a Sephadex G-75 column, then isoelectric focusing to give two protein peaks [Nordberg et al. Biochem J 126 491 1972]. 

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. 

Bier, M Mosher, RA Palusinski, OA, Computer Simulation and Experimental Validation of Isoelectric Focusing in Ampholine-Free Systems, Journal of Chromatography 211, 313, 1981. Bier, M Palusinski, OA Mosher, RA Saville, DA, Electrophoresis Mathematical Modeling and Computer Simulation, Science 219, 1281, 1983. 

Palusinski, OA Allgyer, TT Mosher, RA Bier, M Saville, DA, Mathematical Modeling and Computer Simulation of Isoelectric Focusing with Electrochemically Defined Ampholytes, Biophysical Chemistry 13, 193, 1981. 

Rodbard, D Chrambach, A, In Electrophoresis and Isoelectric Focusing in Polyacrylamide Gels Allen, RC Mauer, HR, eds. Walter E Gruyter New York, 1974 28. 

Figure 4-5. Two-dimensional lEF-SDS-PAGE.The gel was stained with Coomassie blue. A crude bacterial extract was first subjected to isoelectric focusing (lEF) in a pH 3-10 gradient. The lEF gel was then placed horizontally on the top of an SDS gel, and the proteins then further resolved by SDS-PAGE. Notice the greatly improved resolution of distinct polypeptides relative to ordinary SDS-PAGE gel (Figure 4-4).

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. 

Isoelectric focusing of transferrin is a useful biochemical test for assisting in the diagnosis of these conditions truncation of the oligosaccharide chains of this protein alters its isolectric focusing pattern... 

Abnormalities of the glycosylation of transferrin occur in the congenital disorders of glycosylation (Chapter 47) and in chronic alcohol abuse. Their detection by, for example, isoelectric focusing is used to help diagnose these conditions. 

Epiactis prolifera. Extracts of this relatively common species contain two toxins, epiactolysins A and B (13). They have the same molecular weight, namely about 19,500, but differ in pi values as determined by isoelectric focusing. Both lack... 

Hb-A2 can also be quantitated by electrophoresis. The most accurate procedures are starch block electrophoresis, cellulose acetate electrophoresis, and isoelectric focusing. 

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