Isoelectric focusing, capillary

Capillary isoelectric focusing (cIEF) is used to separate zwitterionic compounds, such as peptides and proteins, on the basis of isoelectric point. The separation occurs as a result of a pH gradient that is established inside the capillary. It is important in cIEF to use coated capillaries to reduce or eliminate the EOF and to minimize solute adsorption if the EOF is too swift, it could sweep the analytes past the detector before the separation is complete. 

Two big questions remain How do we establish and maintain a pH gradient across the column while the amphoteric analytes are migrating to their focused halt points, and how then do we move the separated and focnsed bands past a detector while maintaining the separation that was achieved 

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Manning JM et aJ Normal and abnormal protein subunit interactions in hemoglobins.] Biol Chem 1998 273 19359-Mario N, Baudin B, Giboudeau J Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing. J Chromatogr B Biomed Sci Appl 1998 706 123-Reed W, Vichinsky EP New considerations in the treatment of sickle cell disease. Annu Rev Med 1998 49 46l. 

CE was recently used for anthocyanin analysis because of its excellent resolution. This technique has different modes capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), micellar electrokinetic chromatography (MEKC), capillary electrochromatography (CEC), capillary isoelectric focusing (CIEE), and capillary isotachophoresis (CITP)."° CZE is the most popular method for anthocyanin... 

Santora, L. C., Krull, I. S., and Grant, K., Characterization of recombinant human monoclonal tissue necrosis factor-alpha antibody using cation-ex-change HPLC and capillary isoelectric focusing, Anal. Biochem., 275, 98,1999. 

Yim, K. W., Fractionation of the human recombinant tissue plasminogen activator (rtPA) glycoforms by high-performance capillary zone electrophoresis and capillary isoelectric focusing, /. Chromatogr , 559, 401, 1991. 

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

G. Hunt, T. Hotaling, and A. Chen. Validation of a capillary isoelectric focusing method for the recombinant monoclonal antibody C2B8, J. Chromatogr. A, 800, 355 (1998). 

Jensen, P. K., Pasa-Tolic, L., Anderson, G. A., Homer, J. A., Lipton, M. S., Bmce, J. E., and Smith, R. D. (1999). Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 71, 2076-2084. 

Tang, Q., Harrata, A. K., Lee, C. S. (1997). Two-dimensional analysis of recombinant E. coli proteins using capillary isoelectric focusing electrospray ionization mass spectrometry. Anal. Chem. 69(16), 3177-3182. 

Zhou, F., Johnston, M. V. (2005). Protein profiling by capillary isoelectric focusing, reversed phase liquid chromatography, and mass spectrometry. Electrophoresis 26(7-8), 1383-1388. 

Chen, J., Balgley, B.M., DeVoe, D.L., Lee, C.S. (2003a). Capillary isoelectric focusing-hased multidimensional concentration/separation platform for proteome analysis. Anal. Chem. 

Chen, J., Gao, J., Lee, C.S. (2003b). Dynamic enhancements of sample loading and analyte concentration in capillary isoelectric focusing for proteome studies. J. Proteome Res. 2, 249-254. 

Chen, J., Lee, C.S., Shen, Y., Smith, R.D., Baehrecke, E.H. (2002). Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis 23, 3143-3148. 

Shimura, K., Kamiya, K., Matsumoto, H., Kasai, K. (2002). Fluorescence-labeled peptide pi markers for capillary isoelectric focusing. Anal. Chem. 74, 1046-1053. 

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. 

Mao, Y., Zhang, X.M. (2003). Comprehensive two-dimensional separation system hy coupling capillary reverse-phase liquid chromatography to capillary isoelectric focusing for peptide and protein mapping with laser-induced fluorescence detection. Electrophoresis 24, 3289-3295. 

Shen, Y., Berger, S.J., Smith, R.D. (2001). High-efficiency capillary isoelectric focusing of protein complexes from Escherichia coli cytosolic extracts. J. Chromatogr. A 914,257-264. 

Wang W, Guo T, Rudnick PA, et al. Membrane proteome analysis of micro-dissected ovarian tumor tissues using capillary isoelectric focusing/reversed-phase liquid chromatography-tandem MS. Anal. Chem. 2007 79 1002-1009. 

Balgley BM, Wang W, Song T, et al. Evaluation of confidence and reproducibility in quantitative proteomics performed by a capillary isoelectric focusing-based proteomic platform coupled with a spectral counting approach. Electrophoresis 2008 29 3047-3054. 

Several modes of capillary electrophoretic separation are available ordinary CE, capillary zone electrophoresis, capillary electrokinetic chromatography, capillary gel electrophoresis, capillary electrochromatography, capillary isota-chophoresis, and capillary isoelectric focusing. The different separation mechanisms make it possible to separate a wide variety of substances depending on their mass, charge, and chemical nature.53... 

Capillary isoelectric focusing (CIEF) is suitable for the separation of amphoteric analyses in a pH gradient. A continuous pH gradient is built up in the column by using ampholytes under a potential field. Amphoteric analyses migrate to the point where their net charge equal to zero and they form stationary and sharply focused zones. 

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