Proteins In Capillary Electrophoresis Research Articles

Free solution capillary electrophoresis of proteins using untreated fused-silica capillaries

Numerous efforts have been made to separate proteins by capillary zone electrophoresis (CZE). The most common optimization techniques are changing the pH of the running buffer, coating the capillary surface with a hydrophilic polymer, or using additives in the sample solution. Surface coatings and solution additives can reduce the adsorption of the protein onto the capillary surface, but they diminish the separation efficiency and the resolution of CZE. This paper reports the successful separation of proteins in a untreated fused-silica capillary by raising the pH of the running buffer and washing between runs with 1.0 M sodium hydroxide. Under these conditions, model proteins and proteins in human serum have been determined by CZE. It is shown that the results from CZE are compatible with those of sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

High-performance capillary electrophoresis of proteins : Sodium dodecyl sulphate—polyacrylamide gel-filled capillary column for the determination of recombinant biotechnology-derived proteins

Fused-silica capillary columns were filled with sodium dodecyl sulfate-polyacrylamide gel and the column effluent was monitored at 214 nm using a commercially available high-performance capillary electrophoresis (HPCE) instrument to separate and rapidly quantify recombinant biotechnology-derived proteins. An excellent linear relationship ( r>0.999) exists between the peak migration time and the molecular weights of reference proteins in the range 10 000–100 000 and 40 000–200 000 dalton by use of the capillary columns filled with acrylamide gel at a T composition of 5% and 3%, respectively. The relative standard deviation (R.S.D.) of the peak migration time is ca. 1%. Theoretical plates of 5 · 10 5–1 · 10 6 per metre are routinely being obtained. Calibrations graphs of peak area versus weight of recombinant biotechnology-derived proteins are linear ( r>0.999) and the proteins may be quantified with an R.S.D. of ca. 3–7%. As little as 50 nmol of a protein may be quantified and an impurity peak of molecular weight ca. 1500 less than that of the parent compound ( ca. 60 000 dalton) may be differentiated by HPCE with a gel-filled capillary column.

Capillary electrophoresis of proteins under alkaline conditions

Successful separations of proteins by capillary electrophoresis in uncoated fused-silica capillaries is limited by adsorption and variable rates of electroendosmosis, which can compromise quantitative accuracy and precision. Operation at extremes of pH to minimize these problems is useful in special cases but is not a general strategy for protein separations. Three alternative strategies are described: use of capillaries coated with a linear hydrophilic polymer, the use of acidic solutions to wash the capillary between runs, and the incorporation of additives into the electrophoresis buffer to minimize adsorption during analysis. Applications of these techniques to protein samples is demonstrated.

Recent advances in capillary electrophoresis of proteins, peptides and amino acids

The current status of high-performance capillary electrophoresis as an analytical separation method for proteins, peptides and amino acids is assessed. Recent advances in suppressing the effects of electroosmotic flow and irreversible adsorption of proteins at the capillary wall are reviewed, together with procedures for optimal separations of peptides and amino acids. The detection aspects emphasize the role of laser-induced fluorescence and capillary electrophoresis/mass spectrometry in high-sensitivity measurements.

Capillary electrophoresis of proteins in buffers containing high concentrations of zwitterionic salts

A method for improving protein separations in capillary zone electrophoresis utilizing high concentrations of zwitterionic buffer additives was examined. Lysozyme and α-chymotrypsinogen A were used as test proteins in untreated fused-silica capillaries in buffers of pH ca 7.0 and 9.0 The zwitterion-containing buffers were compared with buffers containing high inonic salt concentrations and a buffer containing a combination of high ionic salt and high zwitterion concentrations. Over 100 000 theoretical plates were obtained in less that 30 min for both test proteins in a pH 7 buffer containing both trimethylglycine and potassium sulfate. The advantages and disadvantages of this technique compare with those of other methods used to prevent protein adsorption are discussed.