Effects of increased voltage on resolution in preparative isoelectric focusing of myoglobin varia

Abstract

IEF is a powerful technique which separates proteins and other amphoteric solutes in a pH gradient according to their pI's. The current work evaluates the effect on resolution of increasing electric fields in a novel preparative, vortex-stabilized electrophoresis device. In shallow gradients spanning one pH unit, the variants of myoglobin were separated at applied voltages from 10 to 15 kV. Digital imaging of these separations indicated a 20% reduction in bandwidth and a 60% increase in resolution as the electric field strength is varied across this range. These results were confirmed by IEF-PAGE and ion-exchange chromatography.

Prediction of the location of stationary steady‐state zone positions in counterflow isotachophoresis performed under constant voltage in a vortex‐stabilized annular column

“…The sample train then began moving toward its steady-state position and arrived there approximately 5 h after the start of the experiment. Considering the fact that separation times decrease as applied voltage is increased, this length of time is comparable to preparative IEF experiments performed in the same apparatus, where hemoglobin was focused over a 90 min period at 10 kV [29]. Figure 2 shows a photograph of the contiguous protein zones at two steady-state positions.…”

On-Line Optical Fiber Detection in a Preparative Free-Flow Electrofocusing Apparatus

“…Previous experimentation has demonstrated the performance characteristics of the vortex-stabilized separation annulus (1,29). In this experiment, only pH gradient linearity and stability is examined to illustrate IEF performance.…”

“…The vortex-stabilized separation chamber is a flexible preparative free-flow electrofocusing apparatus that has been used in many applications including continuous fractionation of enantiomer pairs (28), ampholyte-free IEF using defined binary buffers (6), IEF of isoforms (1), and IEF at increased electric field strengths (29). All of these applications could benefit from an online detection system.…”

Protein separation using preparative‐scale dynamic field gradient focusing

“…Separating and focusing proteins by their electrophoretic mobilities in native buffers using DFGF avoids the precipitation problem inherent in focusing proteins at their pI where they are often least soluble. Enhancing the resolution of IEF separations requires harvesting the samples of interest and re-running them in a shallower gradient [15], but the resolution of DFGF separations is adjustable during the run [2]. For example, the resolution between two proteins can be increased by changing the buffer's pH to increase the difference in mobility between two proteins [2], or the slope of electric field gradient could be decreased to move the focal points of the two proteins further apart [2].…”