Capillary SDS-gel electrophoresis

Capillary SDS-gel electrophoresis is a rapid automated separation and characterization technique for protein molecules and is contemplated as a modern instrumental approach to sodium dodecylsulfate-polyacrylamide slab-gel electrophoresis (SDS-PAGE). Size separation of SDS-protein complexes can be readily attained in coated capillaries filled with cross-linked gels or non-cross-linked polymer networks. Figure 9 depicts one of the early applications of the technique for the analysis of a standard protein test mixture ranging in size from 14.2 to 205 kDa. 

Capillary SDS-gel electrophoresis has proved to be a very important separation tool for rapid molecular-weight estimation and purity check of recombinant proteins in the modern biotechnology industry. A comprehensive review of capillary gel electrophoresis of proteins was published by Guttman in 1996 [40]. Since then, the field has moved toward biomedical and biotechnology applica-... 

Figure 9 Capillary SDS-gel electrophoresis trace of a protein testmixture (1) 0-lactalbumin, MW 14,200 (2) carbonic anhydrase, MW 29,000 (3) ovalbumin, MW 45,000 (4) bovine serum albumin, MW 66,000 (5) phosphorylase B, MW 97,400 (6) P-galactosidase, MW 116,000 (7) myosin, MW 205,000 (OG) tracking dye Orange-G. Conditions E = 300 V/cm Injection, 100-ng protein mix detection, 214 nm. (Reproduced with permission from Ref. 162.)...

Guttman, A., Horvath, J., and Cooke, N., Influence of temperature on the sieving effect of different polymer matrices in capillary SDS gel electrophoresis of proteins, AnoZ. Chem., 65, 199,193. Giordano, B. C., Horsman, K. M., Burgi, D. S., Eerrance, J. R, and Landers, J. R, Method for determining intracapiUary solution temperatures application to sample zone heating for enhanced fluorescent labeling of proteins. Electrophoresis, 28, 714, 2007. 

Non-denaturing gel electrophoresis Denaturing (SDS) gel electrophoresis Two-dimensional electrophoresis Capillary electrophoresis Peptide mapping HPLC (mainly RP-HPLC)... 

Figure 17 SDS-capillary gel electrophoresis of protein standards. Separation conditions 27 cm x 50 pm i.d. uncoated capillary 888 V/cm gel eCAP 200. (From Bene-dek, K. and Guttman, A., ]. Chromatogr., 680, 375, 1994. With permission.)...

The various types of capillary electrophoresis are performed either in free solution or in gels. The choice of method depends on the nature of the sample and the analytical objective but capillary gel electrophoresis, including iso-electric focusing and SDS electrophoresis, is particularly useful for protein applications. 

The retardation of the SDS-protein complexes in capillary gel electrophoresis is a function of the separation polymer concentration (P) and the retardation coefficient (KR) ... 

Surfactants are amphophilic molecules, which consist of a hydrophobic carbohydrate part and a hydrophilic head group. In capillary zone electrophoresis (CZE), different types, i.e., anionic, cationic, but also neutral, tensides are employed. The ability of such molecules to interact with ionic and nonionic species has been used in ion chromatography and, in particular, in SDS-poly-(acrylamide) gel electrophoresis (PAGE) (15). 

It has often been realized that the separation efficiency in the competitive CEIA mode is favorable compared to the noncompetitve mode, especially if free and bound antibodies are utilized. Ou and co-workers presented a relatively simple approach to solve this problem (15,16). They used nondenaturing SDS capillary gel electrophoresis (CGE) to analyze anti-bovine serum albumin (BSA) antibodies with UV detection. 

Chemical purity. The purity of the protein product is analysed in comparison with a reference preparation by a suitable method, such as liquid chromatography, capillary electrophoresis or sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). 

Klyushnichenko, V., Tishkov, V. andKula, M.R. (1997) Rapid SDS-Gel capillary electrophoresis for the analysis of recombinant NADP(+)-dependent formate dehydrogenase during expression in Escherichia coli cells and its purification. J... 

Edman-based protein sequencing, immunostaining, SDS-capillary gel electrophoresis for protein quantification at picomolar range... 

Application 2 Capillary Gel Electrophoresis. Recently, Karger and co-workers demonstrated the use of polyacrylamide gel-filled capillaries to separate peptide/protein (SDS PAGE) Qfi) and oligonucleotide mixtures (12,22) by capillary electrophoresis. This mode of CE operation may prove to couple well with on-line radioisotope detection. The results of several preliminary capillary electrophoresis separations using gel-filled capillaries and on-line radioisotope detection using the coincidence unit described here are presented below. 

The separation principle in capillary gel electrophoresis (CGE) is the same as that of slab gel electrophoresis. Most often CGE is used in a denaturing mode with the incorporation of SDS and is referred to as SDS-CGE. As such, separation is based on the protein s molecular mass and, due to the sieving mechanism of the gel, smaller proteins migrate past the detector first. The use of a gel material and SDS decreases the EOF and eliminates protein adsorption to the capillary walls further ensuring that migration is based on molecular mass. This precludes the need for additives and coated capillaries. 

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