Isoelectric focusing, electrophoresis

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. 

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). 

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

CE is a family of techniques similar to those found in conventional electrophoresis zone electrophoresis, displacement electrophoresis, isoelectric focusing (IEF), and sieving separations. Other modes of operation unique to CE include micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC). 

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). 

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. 

Osterman LA (1984) Methods of protein and nucleic acid research, vol. 1 Electrophoresis, isoelectric focusing, ultracentrifugation. Springer, Berlin, p. 284... 

Schmitt, R, Poiger, T., Simon, R., Garrison, A. W., Freitag, D., and Kettrup, A. (1997b). Simultaneous ionization constants and isoelectric points determination of 12 hydroxy-s-triazines by capillary zone electrophoresis (CZE) and capillary electrophoresis isoelectric focusing (CIEF). Anal. Chem. 69,2559-2566. 

The separation scientist with experience gained from a LC background may tend to limit the modes of electrochromatography to reversed phase (RP), normal phase, ion-exchange and, maybe, size-exclusion. Analysts from an electrophoretic background typically use the term "CE" in a much broader sense to include the main modes of capillary zone electrophoresis, micellar electrokinetic chromatography, capillary gel electrophoresis, isoelectric focusing and isotachophoresis. 

Figure 8.9. Resolution of proteins from heart muscle by 2D electrophoresis isoelectric focusing (IEF) x gel electrophoresis (GE). Sample size, lmg wet weight silver stain. (Courtesy of E. Jellum, University of Oslo.)...

The most distinguishing phenomenon in capillary electrophoresis is whether the experiments are performed in the absence or in the presence of electroos-motic flow (EOF), (see Chapter 6 for details on EOF). Unlike other types of capillary electrophoresis, isoelectric focusing can be performed under both modes. Since the experimental and theoretical principles governing these modes of CIEF are different, they will be discussed separately. 

BAP measurement methods have used heat inactivation, chemical inhibition, electrophoresis, isoelectric focusing, lectin precipitation and chromatography, HPLC, and other approaches, singly and in combination. Conventional elec-trophoresis provides qualitative, not quantitative, detection because of the overlapping migration of isoforms and isoenzymes. Heat inactivation and chemical inhibition with urea, hphenylalanine, 1-homoarginine, or levamisole have been used with some success. Internal standards of ALP isoenzymes have been used to increase assay precision. 

Other equally important methods are the electrophoretic techniques, such as polyacrylamide gel electrophoresis, isoelectric focusing, Western blots, combined electrophoresis, and isoelectric focusing (two-dimensional electrophoresis). Bioactivity methods are other key methods used in biotechnology product development. These include in vivo whole animal bioassay, cell culture bioassay, immunoassay, and biochemical assay. Many references and several textbooks are available in many industrial and academic libraries to provide additional and up-to-date information. 

Gll. GSrg, A., Postel, W., and Westermeier, R., Ultra-thin-layer horizontal electrophoresis, isoelectric focusing and protein mapping in polyacrylamide gels on cellophane. In Electrophoresis 79 (B. J. Radola, ed.), pp. 67-78. Walter de Gruyter, Berlin, 1980. 

Ionic charge pH precipitation Ion exchange chromatography Gel electrophoresis Isoelectric focusing... 

See alsa Electrophoresis Isoelectric Focusing. Ion-Selective Eiectrodes Glass. pH. Quality Assurance Instrument Calibration. 

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