Electrophoresis capillary isoelectric focusing

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 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. 

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). 

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. 

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. 

ID Separation of Proteins There has been considerable research devoted to tbe development of nricioflu-idic platforms capable of performing small-scale protein separations. In addition to diffusion based methods [3], nearly every type of electrokineticaUy driven separation has been demonstrated in a cbip-based platform, including free-flow electrophoresis, capillary electrophoresis, capillary gel electrophoresis, isoelectric focusing (lEF), micellar electrokinetic chromatography and capillary electrochemical chromatography. 

CE was recently used for anthocyanin analysis because of its excellent resolution. This technique has different modes capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEE), and capillary isotachophoresis (CITP)."° CZE is the most popular method for anthocyanin... 

Zhu, M., Hansen, D.L., Burd, S., and Gannon, F., Factors affecting free zone electrophoresis and isoelectric focusing in capillary electrophoresis, /. Ckromatogr., 480, 311, 1989. 

Hjerten, S., Isoelectric focusing in capillaries, in Capillary Electrophoresis, Gross-man, P. D. and Colburn, J. C. Eds., Academic Press, San Diego, 1992. 

Kilar, E and Hjerten,S., Fast and high resolution analysis of human serum transferrin by high performance isoelectric focusing in capillaries, Electrophoresis, 10, 23, 1989. 

Yim, K. W., Fractionation of the human recombinant tissue plasminogen activator (rtPA) glycoforms by high-performance capillary zone electrophoresis and capillary isoelectric focusing, /. Chromatogr , 559, 401, 1991. 

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. 

Three bacterial species (E. coli, P. putida, and S. rubidae) were separated on isoelectric focusing in methylcellulose coated capillaries, and three bacterial species (P. fluorescens, E. aerogenes, and M. luteus) and the yeast S. cerevisae, were separated by capillary electrophoresis in the presence of polyethylene oxide.101 The polymers served to minimize adsorption to the walls without causing cellular lysis. 

Zhou, F., Johnston, M. V. (2005). Protein profiling by capillary isoelectric focusing, reversed phase liquid chromatography, and mass spectrometry. Electrophoresis 26(7-8), 1383-1388. 

Chen, J., Lee, C.S., Shen, Y., Smith, R.D., Baehrecke, E.H. (2002). Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis 23, 3143-3148. 

High-efficiency separations of FQ-labeled proteins are only achieved in the presence of an anionic surfactant, such as SDS. As a result, capillary isoelectric focusing is not useful for the analysis of these proteins. Instead, we employ capillary sieving electrophoresis and micellar electrokinetic capillary chromatography for our two-dimensional electrophoresis. 

Mao, Y., Zhang, X.M. (2003). Comprehensive two-dimensional separation system hy coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection. Electrophoresis 24, 3289-3295. 

Balgley BM, Wang W, Song T, et al. Evaluation of confidence and reproducibility in quantitative proteomics performed by a capillary isoelectric focusing-based proteomic platform coupled with a spectral counting approach. Electrophoresis 2008 29 3047-3054. 

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