Electrophoresis cell capillary

Hu, S., Michels, D.A.,Fazal,M.A., Ratisoontom, C., Cunningham, M.L., Dovichi,NJ. (2004). Capillary sieving electrophoresis/micellarelectrokinetic capillary chromatography for two-dimensional protein fingerprinting of single mammalian cells. Anal. Chem. 76,4044-4049. 

FIG. 12.10 Velocity profiles in electrophoresis cells (a) velocity (as time-1) of Klebsiella aerogenes particles as a function of their location in a rectangular electrophoresis cell (redrawn with permission from A. M. James, in Surface and Colloid Science, Vol. 11 (R. J. Good, and R. R. Stromberg, Eds.), Plenum, New York, 1979) (b) location of the surface of zero liquid velocity in a cylindrical capillary. 

Munce, N.R., Li, J., Herman, P.R., Lilge, L., Microfabricated system for parallel single-cell capillary electrophoresis. Anal. Chem. 2004, 76, 4983-4989. 

Hu, S., Zhang, L., Krylow, S. and Dovichi, N.J. (2003a) Cell cycle-dependentprotein fingerprint form a single cancer cell i mage cytometry coupled with single-cell capillary sieving electrophoresis. Anal. Chem. 75,3495-3501. 

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. 

Cruz, L. et al.. Nitrite and nitrate levels in individual molluscan neurons Single-cell capillary electrophoresis analysis, J. Neurochem., 69, 110, 1997. 

FIG. 2 Schematic diagram of the capillary electrophoresis cell for the Zetasizer 3. (From Ref 7.)... 

Hu, S., Zhang, L., Newitt, R., Aebersold, R., Kraly, J. R., Jones, M., Dovichi, N. J. Identification of proteins in single-cell capillary electrophoresis fingerprints based on comigration with standard proteins. Anal. Chem. 2003, 75, 3502-3505. 

Khan, H. A. The effect of DNA labeling with the fluorescent dyes RllO and R6G on genotype analysis using capillary electrophoresis. Cell. Mol. Biol. Lett. 2005,10, 247-253. 

Limits of detection become a problem in capillary electrophoresis because the amounts of analyte that can be loaded into a capillary are extremely small. In a 20 p.m capillary, for example, there is 0.03 P-L/cm capillary length. This is 1/100 to 1/1000 of the volume typically loaded onto polyacrylamide or agarose gels. For trace analysis, a very small number of molecules may actually exist in the capillary after loading. To detect these small amounts of components, some on-line detectors have been developed which use conductivity, laser Doppler effects, or narrowly focused lasers (qv) to detect either absorbance or duorescence (47,48). The conductivity detector claims detection limits down to lO molecules. The laser absorbance detector has been used to measure some of the components in a single human cell (see Trace AND RESIDUE ANALYSIS). 

Several additional instrumental techniques have also been developed for bacterial characterization. Capillary electrophoresis of bacteria, which requires little sample preparation,42 is possible because most bacteria act as colloidal particles in suspension and can be separated by their electrical charge. Capillary electrophoresis provides information that may be useful for identification. Flow cytometry also can be used to identify and separate individual cells in a mixture.11,42 Infrared spectroscopy has been used to characterize bacteria caught on transparent filters.113 Fourier-transform infrared (FTIR) spectroscopy, with linear discriminant analysis and artificial neural networks, has been adapted for identifying foodbome bacteria25,113 and pathogenic bacteria in the blood.5... 

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. 

This instrumentation has exquisite sensitivity, which allows the analysis of single cancer cells (Hu et al., 2004). Our earlier work employed slow separation conditions and a rather primitive photodetection system. Our current system takes roughly 1 h to complete the two-dimensional capillary electrophoresis separation and employs state-of-the-art photodetectors. 

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