Capillary zone electrophoresis in agarose gels using absorption imaging detection

Abstract

A simple home-built electrophoretic unit and a commercially available charge-coupled device (CCD) camera with image acquisition and analysis software were used to study the separation process in zone electrophoresis experiments in 4 cm long, round capillaries (inside diameter 0.2 mm). Several capillaries could be investigated simultaneously. The absorption imaging system was used not only to follow the course of the separation but also to study the interaction between biologically active substances (proteins, detergents, enzymes and-substrate). Since the system allows visual on-line observation of the separation one can rapidly decide when the analysis is finished, which often shortens the analysis time. The electrophoresis method presented is suitable also for preparative runs, since direct visualizations of a solute zone allows it to be excised and then used for further studies.

Whole-column imaging capillary electrophoresis of proteins with a short capillary

“…Another method was to fill the whole short capillary with the agrose gel, as in previous experiments [5]. Figure 3 shows the CE separation of the proteins in an agarose gel-filled 4 cm capillary with different injection times and different pH.…”

“…In previous experiments, it was demonstrated that wholecolumn imaging CE could be used for studying interaction between proteins and biologically active substances such as the detergent CTAB [5]. Here, protein binding or conjugation reaction between cytochrome c and surfactant SDS was investigated.…”

“…In our experiments, a short capillary (3-6 cm) was used as a separation capillary. In addition to CIEF, the wholecolumn imaging detection was also applied to capillary zone electrophoresis (CZE) in an agarose gel-filled short capillary [5]. Our CIEF experimental results have proved that the whole-column imaging detection permits the direct observation of the IEF dynamics and accelerates the analysis speed greatly.…”

“…Recently, many CE experiments have been performed in a microchannel fabricated in a chip [1][2][3][4] or a short capillary [5,6] with a length of several centimeters. Conventional CE is carried out in a long capillary (from * 10 to 100 cm).…”

“…Three types of whole-column optical imaging detection namely refractive index gradient [10][11][12], fluorescence [14] and absorbance [15] imaging detection have been established. The experimental results showed that the whole-column imaging CZE could be used for study of interaction between protein and biologically active sub-stances such as detergent cetyltrimethylammonium bromide (CTAB) [5]. In addition to CIEF, the wholecolumn imaging detection was also applied to capillary zone electrophoresis (CZE) in an agarose gel-filled short capillary [5].…”