Isoelectric focusing chromatography

Methods for AMY isoenzymes based on electrophoresis, ion-exchange chromatography, isoelectric focusing, selective inhibition of the S-AMY by a wheat germ inhibitor, immunoprecipitation by a monoclonal antibody, and immunoinhibition have been introduced. However, only the methods based on the selective isoenzyme inhibition by monoclonal antibodies have shown sufficient precision, reliability, practicability, and analytical speed to allow the introduction of the P-AMY determination in clinical practice. 

Fluorescence bound to albumin, extractable with charcoal treatment under acidic conditions, soluble in water and polar alcohols, diminished at pH <3 or >12 Gel chromatography, isoelectric focusing, protein electrophoresis, thin-layer chromatography, extraction with charcoal and alcohol... 

In microfluidic-based systems, material is transported within microstructures (of typical dimensions of 10-500 pm) where separations, reactions, and other processes occur. Focus has been on the realization of the traditional separation techniques (electrophoresis, chromatography, isoelectric focusing, etc.) and reactions in the microchip format. The principles of separation, as in the conventional formats, are based on differences in mass and charge (thus mobility) and partitioning between phases. However, advantages associated with the small dimensions provide superior performance. For example, the higher surface to volume ratio arising from the smaller dimensions results in lower heat and mass transfer resistances and thus an improved performance. 

For this reason, glycoproteins must first be isolated from the biological matrix by dialysis, preparative chromatography, isoelectric focusing, and so forth or by a combination of several methods. For a structural determination, degradation steps such as a site-specific proteolysis (e.g., with trypsin), removal of oligosaccharides from the polypeptide (by an enzymatic hydrolysis or hydrazine... 

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]. 

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. 

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... 

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). 

The column methods are much faster and are automated so that a much larger number of samples can be processed per unit time. An example of this technology, described in more detail in Chapter 10 by Lubman and coworkers, is shown in Figure 1.2, where the first dimension is from a chromatofocusing column, which gives separations in pI much like isoelectric focusing, only here the p/ axis is in bands instead of continuous pI increments. The second dimension is by reversed-phase liquid chromatography (RPLC). 

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. 

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. 

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. 

This effect is illustrated in Fig. 17.1. Multidimensional chromatography separations can be done in planar systems or coupled-column systems. Examples of planar systems include two-dimensional thin-layer chromatography (TLC) (Consden et al., 1944 Grinberg et al., 1990), where successive one-dimensional TLC experiments are performed at 90° angles with different solvents, and 2D electrophoresis, where gel electrophoresis is run in the first dimension followed by isoelectric focusing in the second dimension (O Farrell, 1975 Anderson et al., 1981 Celis and... 

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