Capillary electrophoresis, free solution proteins

Bullock, J.A. and Yuan, L.-C., Free solution capillary electrophoresis of basic proteins in uncoated fused silica capillary tubing, ]. Microcol. Sep., 3,241,1991. 

P. D. Grossman, J. C. Colburn, H. H. Lauer, R. G. Nielsen, R. M. Riggin, G. S. Sittampalam and E. C. Rickard, Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides . Anal. Chem. 61 1186-1194 (1989). 

Lee, K.-J. and Heo, G. S., Free solution capillary electrophoresis of proteins using untreated fused-silica capillaries, ]. Chromatogr., 559, 317, 1991. 

Karim, M.R., Shinagawa, S., Takagi, T. (1994). Electrophoretic mobilities of the complexes between sodium dodecyl sulfate and various peptides or proteins determined by free solution electrophoresis using coated capillaries. Electrophoresis 15, 1141-1146. 

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. 

Gel electrophoresis provides a simple method for separating complex protein mixtures. Because proteins are visualized using stains that may not be linearly incorporated in the gel, the intensity of the stained bands may be poorly correlated with the amount of protein. For this reason, gel electrophoresis is at best a semiquantitative technique capable of generating relative purity results. In CE, separations are commonly performed in free solution, i.e., in the absence of any support such as gel matrices. This allows the replacement of the capillary s content in between analyses and therefore the automation of the process. The use of UV-transparent fused-silica capillaries enables direct on-line optical detection of focused protein zones, eliminating the requirement for sample staining. The detection systems available to CE provide true quantitative capabilities. 

VJ Hilser, CD Worosila, E Freire. Analysis of thermal-induced proteins folding/ unfolding transitions using free-solution capillary electrophoresis. Anal. Biochem. 208 125-131 (1993). 

DK Lloyd, S Li, P Ryan. Protein chiral selectors in free-solution capillary electrophoresis and packed-capillary electrochromatography. J Chromatogr A 694 285-296, 1995. 

Capillary zone electrophoresis (CZE), also known as free-solution CE, is the most widely used mode of CE essentially because of its versatility. Protein separation in CZE is based on the differential electrophoretic mobility of the analytes. This mobility is primarily dependent on a protein s size and net charge, the charge-to-mass ratio. Solvent properties that influence the size and charge of a protein include pH, ionic strength, viscosity, and dielectric constant.67 Manipulation of these properties, most notably pH, dictates the selectivity in CZE. Maximizing the charge difference between two proteins via pH modification optimizes their separation. 

Bishop, R.T., Turula, V.E., and de Haseth, J.A. Study of Conformational Effects on Reversed-Phase Chromatography of Proteins with Particle Beam LC/FT-IR Spectrometry and Free Solution Capillary Electrophoresis (1996) Anal. Chem., in press. 

Even though free-solution CE is most commonly used for neuropeptides and neuroproteins, other forms of CE have also been employed. For instance, as an alternative to conventional slab-gel electrophoresis, a method using sodium dodecyl sulfate (SDS) capillary gel electrophoresis was developed. It was applied to low-molecular-mass proteins (j8-trace protein, ft-microglobulin, -trace protein, and myelin basic protein) in cerebrospinal fluid [4], Advantageous features of capillary gel electrophoresis over slab-gel electrophoresis are compatibihty with small sample volumes, shorter analysis times, and more accurate quantihcation of the analytes. 

Dubin et al. used capillary electrophoresis to separate the complexes of proteins with synthetic polyanions from the free protein in the complex solution and to determine the complex composition. The binding isotherms were studied in relation to the pH value [70], the chain length of the poly-... 

Meagher, R. J., Won, J. I., and Barron, A. E. Sequencing of DNA by free-solution capillary electrophoresis using a genetically engineered protein polymer drag-tag. Submitted (2006). 

Grossman, P.D. et al. Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides, AnaZ. Chem., 61, 1186, 1989. 

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. 

All these separation modes can be performed in different instrumental formats. The first electrophoretic separations of proteins were performed by Tiselius in a free solution in a U-tube macrocuvette, allowing refraction index detection of boundaries of protein zones. Soon it was found out that the separation would be more efficient if performed in anti-convective stabilizing medium thus electrophoresis started to be performed in different carriers (paper, acetyl cellulose membrane) or in gel media (starch, agar, agarose, polyacrylamide (PAA)). In the last two decades electrophoresis has reverted to the free solution, stabilized by the anticonvective capillary effect of microbore (inner diameter (i.d.) <100pm)... 

Figure 7 CZE separation of hGH and its derivatives. CZE performed in FS capiiiary (i.d. 50 pm, totai iength 105cm, effective iength (to detector) 81.5cm BGE lOmmoii" Tricine, 5.8mmoii morphoiine, 20mmoii NaCi, pH8.0 eiectric fieid intensity 300Vcm current 20pA, temperature 24°C. 1, hGH 2, (desamido-149)- and (desamido-152)-hGH 3, (didesamido-149-152)-hGH. Mesityl oxide, electroosmotic fiow marker. (Reprinted with permission from Grossman PD, Coiburn JC, Lauer HH, et al. (1989) Application of free-solution capillary electrophoresis to the anaiyticai scaie separation of proteins and peptides. Analytical Chemistry 61(11) 1186-1194 American Chemical Society.)...

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