Isoelectric focusing and

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. 

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. 

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. 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

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. 

Essader, A.S., Cargile, B.J., Bundy, J.L., Stephenson, J.L., Jr. (2005). A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics. Proteomics 5, 24—34. 

Ruchel, R. (1977). Two-dimensional micro-separation technique for proteins and peptides, combining isoelectric focusing and gel gradient electrophoresis. J. Chromatogr. 132, 451 168. 

KLOSE, J., Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals, Humangenetik, 1975, 26,231-243. 

Two variations of the basic technique are isoelectric focusing and immuno-electrophoresis. The former offers improved resolution and sharper bands in the separation of weak acids, weak bases and ampholytes through the use of pH and density gradients superimposed along the potential gradient. The latter employs specific antigen-antibody interactions (Chapter 10) to visualize the separated components of serum samples. 

Describe the following electrophoresis, zone electrophoresis, paper electrophoresis, gel electrophoresis, isoelectric focusing, and capillary electrophoresis. 

This introduction was intended to merely provide basic concepts and principles of electrophoretic separation. It should be noted that a wide variety of electrophoretic techniques, such as isoelectric focusing and two-dimensional gel... 

Purity and homogeneity of the purified protein is assessed by macromolecular exclusion chromatography, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The later technique, developed by Karas and Hillenkamp, ionizes and separates proteins on the basis of their mass-to-charge ratio (Karas and ffillenkamp, 1988). 

Capillary zone electrophoresis (CZE) is the most common electrophoretic separation technique due to its simplicity of operation and its flexibility. It is the standard mode for drug analysis, identification of impurities, and pharmacokinetic studies. Other separation modes, such as capillary isotachopho-resis (CITP), micellar electrokinetc chromatography (MEKC), capillary electrochromatography (CEC), capillary gel electrophoresis (CGE), capillary isoelectric focusing, and affinity capillary electrophoresis (ACE), have then-advantages in solving specific separation problems, since the separation mechanism of each mode is different. 

RG Nielsen, EC Rickard, PF Santa, DA Shakarnas, GS Sittampalam. Separation of antibody-antigen complexes by capillary zone electrophoresis, isoelectric focusing and high-performance size-exclusion chromatography. J Chromatogr 539 177-185, 1991. 

Ceruloplasmin (from human blood plasma) [9031-37-2]. Purified by precipitation with polyethylene glycol 4000, batchwise adsorption and elution from QAE-Sephadex, and gradient elution from DEAE-Sepharose CL-6B. Ceruloplasmin was purified 1640-fold. Homogeneous on anionic polyacrylamide gel electrophoresis (PAGE), SDS-PAGE, isoelectric focusing and low speed equilibrium centrifugation. [Oestnuizen AB 146 1 1985]. 

Combining isoelectric focusing and SDS electrophoresis sequentially in a process called two-dimensional... 

Trieu-Cuot, P. and Gripon, J. C. 1982. A study of proteolysis during Camembert cheese ripening using isoelectric focusing and two-dimensional electrophoresis. J. Dairy Res. 49, 501-510. 

Figure l shows the gold stain of a section of a 2D gel of beef heart mitochondria proteins separated by isoelectric focusing and then by SDS-PAGE, and transferred to nitrocellulose. The filter was then probed with antibody specific to the 49-kDa iron sulfur protein of NADH CoQ reductase. The blot was photographed with (Fig. 2) and without (Fig. 3) a red filter following development with 4-chloro-1 -naphthol. The position of the 49-kDa protein can then be determined by back reference to Fig. 1 (see arrow). 

HK Mayer, D Heidler, C Rockenbauer. Determination of the presence of the percentages of cows , ewes and goats milk in cheese by isoelectric focusing and cation-exchange HPLC of y- and para-/c-caseins. Int Dairy J 7 619-628, 1997. 

Moio, L., Chianese, L., Rivemale, M., and Addeo, F. (1992). Fast detection of bovine milk in Roquefort cheese with PhastSystem by gel isoelectric focusing and immunoblotting. Lait 72, 87-93. 

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