One-dimensional capillary electrophoresis

FIGURE 15.1 One-dimensional capillary electrophoresis separation of a protein homogenate prepared from the hTERT cell line. Both separations were preformed in 30 pm ID, 145 pm OD, 20 cm long capillaries at 20,000 V. (a) Micellar electrokinetic chromatography performed with a 100 mM CHES, 100 mM Tris, and 15 mM SDS buffer at pH 8.7. Sample is electro-kinetically injected with 0.25 kV for 1 s (b) Capillary sieving electrophoresis performed in 5% Dextran (513 kDa), 100 mM CHES, 100 mM Tris, 3.5 mM SDS, pH 8.7. 

Chemical Cytometry of Biogenic Amines and Proteins Using One-Dimensional Capillary Electrophoresis... 

Figure 21.4 presents a one-dimensional capillary sieving electrophoresis (CSE) analysis of the proteins from a human primary T-cell. In this separation, the cell was injected into the capillary, lysed, and primary amines were labeled with FQ. Components were separated by CSE. The electrophero-gram consists of a set of 25 peaks, some of which are extraordinarily sharp for example, the component that migrates at 14.2 min generates a peak with 2.8 million theoretical plates (7.7 million plates/m), whereas most peaks generate a few hundred thousand theoretical plates. 

A new topic is now included Chapter 20 about quahty assurance. Part of it could be found before in chapter 19 but now the subject is presented much broadly and independent of Analytical HPLC . Two chapters in the appendix were updated and expanded by Bruno E. Lendi, namely the ones about the instrument test (now chapter 25) and troubleshooting (now chapter 26). Some new sections were created 1.7, comparison of HPLC with capillary electrophoresis 2.11, how to obtain peak capacity 8.7, van Deemter curves and other coherences 11.3, hydrophilic interaction chromatography 17.2, method transfer 18.4, comprehensive two-dimensional HPLC 23.3, fast separations at 1000 bar 23.5, HPLC with superheated water. In addition, many details were improved and numerous references added. 

A cell contains a myriad of proteins. The individual proteins are separated and identified to understand the nature of different proteins and their structural and functional relationships. Proteins are isolated by a variety of means because they have a diverse set of properties. A multidimensional approach is more suitable to separate them than a single approach, as outlined in Chapter 1. Such a multidimensional approach must address the problems concerning their resolution, high throughput, automation, and adaptability to analysis by mass spectrometry. One of the most important ways they are separated is by electrophoresis, including the two-dimensional (2D) gel electrophoresis and capillary electrophoresis. After their separation by these methods, they are identified by use of a mass spectrometry. These methods are described in this chapter. 

IEF is the conventional method for protein separation in two-dimensional (2D) polyacrylamide gel electrophoresis and is considered one of the most powerful techniques available for separating proteins. However, IEF in gels is a time-consuming technique. Hjerten and Zhu, therefore, adapted IEF to fused-silica capillaries, so-called capillary IEF (cIEF), to minimize analysis times. No gels are used in cIEF instead, the capillary is filled with ampholytes in free solution to create the pH gradient. This technique is also called liquid-phase IEF. Additional advantages of cIEF are the potential for system automation and the possibility of on-line detection. 

Capillary nonequilibrium pH gradient gel electrophoresis in conjunction with mini slab polyacrylamide electrophoresis is a rapid, cost-effective, and easy-to-handle system for the two-dimensional analysis of complex protein mixtures in one day. 

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