Capillary electrophoresis sieving

Widhalm et al. (1991) reported the use of noncrosslinked polyacrylamide for protein separation in fused silica capillaries. This matrix has low viscosity and can be replaced between separations, greatly facilitating automation of the separation. A wide range of noncrosslinked polymers has been used for size-based protein separations. Noncrosslinked polymers do not form a gel, and it is inappropriate to refer to this separation as gel electrophoresis. A number of names have been used for the method. In an effort to standardize nomenclature, IUPAC has used the term capillary sieving 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. 

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. 

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. 

Michels, D.A., Hu, S., Damhrowitz, K.A., Eggertson, M.J., Lauterhach, K., Dovichi, NJ. (2004). Capillary sieving electrophoresis-micellar electrokinetic chromatography fully automated two-dimensional capillary electrophoresis analysis of Deinococcus radiodurans protein homogenate. Electrophoresis 25, 3098-3105. 

Capillary gel electrophoresis (CGE) — A special case of - capillary sieving electrophoresis in which the capillary is filled with a cross-linked gel. 

Capillary sieving electrophoresis (CSE) — An electrophoresis technique, in which analytes are separated in a capillary, containing a sieving medium (e.g., an entangled polymer network) in the -> supporting (background) electrolyte. The separation is based on differences in size and shape of the charged analytes. 

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. 

SDS-puIIuIan (capillary sieving electrophoresis) followed by MECC Formic acid (1 M, pH 1.78) for CZE followed by 125 mM ammonium fomate (pH4)fortCITP Hios[4iate buffer (50 mM)... 

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. 

Blanco, D. Junco, S. Exposito, Y. Gutierrez, D. Study of various treatments to isolate low levels of cider proteins to be analyzed by capillary sieving electrophoresis. J. Liq. Chromatogr. 2004, 27, 1523-1539. 

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