Recent applications of capillary isoelectric focusing

Abstract

After the advent of capillary isoelectric focusing (CIEF) in the 80's several approaches have been developed in order to use the technique in routine analyses. The recent years showed an extensive increase in the applications of this technique employing its exceptionally high-resolution power. Methodological improvements, as well as hyphenation with other electrophoretic and chromatographic separation procedures, proved the versatility of CIEF in studies of clinically important proteins, recombinant product, cell lysates and other complex mixtures. The combination of CIEF with mass spectrometry detection is one of the major challenges for studying proteomics. This review collected the recent applications of CIEF including innovations in the experimental setup, remedies for the presence of salts in samples, calibration of the pH gradient, carrier ampholyte-free isoelectric focusing, the progress in micropreparation, two-dimensional separations, etc.

Capillary electrophoresis of proteins 2003–2005

“…In postgenomic era, the importance of protein separations increased significantly. CIEF as the most efficient technique for separation of proteins attracted substantial attention [136]. Easy combination of CIEP and MS was an additional attractive feature for bioanalytical chemists [137,138].…”

Cascaded Free-Flow Isoelectric Focusing for Improved Focusing Speed and Resolution

“…(1) (2) From Equation 1, the focusing resolution, Res, depends on the square root of the electric field, E, mobility μ, the difference in species charge Δz across the channel width w, divided by diffusion constant D. Equation 2 describes the relationship between the characteristic focusing time (t*) and focusing distance as linear with respect to w.…”

Preparative capillary zone electrophoresis using a dynamic coated wide‐bore capillary

“…Using 100-mm id capillaries, loadings of 0.3 mg of each protein have been achieved [12]. However, the mobilization step in CIEF can distort the pH gradient created in the column during the focusing step resulting in poor reproducibility, longer analysis times, and degradation of resolution between the focused zones [11]. Further, proteins with comparable pI cannot be separated using CIEF (estimated resolution is 0.05 pH units [12]).…”

“…Consequently, extensive efforts have been devoted to the development of preparative non-gel-based techniques to facilitate protein isolation. One of the non-gel electrophoretic methods that has been used for preparative protein separations is CIEF [10,11]. Using 100-mm id capillaries, loadings of 0.3 mg of each protein have been achieved [12].…”