The principle of free-flow electrophoresis (FFE) was first introduced by Barrolier (1958) and Hannig (1961). FFE provides a liquid-based separation in three different operating modes: zone electrophoresis-separation of particles (cells, organelles) due to their electrophoretic mobility, isotachphoresis-separation of proteins and peptides in pH step gradient and isoelectric focusing-separation of proteins and peptides due to their isoelectric point. The key feature of FFE technology is as follows. (i) The separation is performed continuously and enables us to obtain as much as hundreds of milligrams or even gram amounts pure substances. (ii) the separation is performed in a thin aqueous film without gels and enables us to collection of matrix-free fraction with high reproducibility. Therefore, FFE technology ensures that the separated samples are compatible with all downstream concentration procedures (e.g. ultrafiltration), whose enrichment allows to visualize less abundant proteins for subsequent 2-DE analysis and separate poor soluble proteins (e.g. membrane proteins) for subsequent SDS-PAGE. Moreover, FFE can be coupled with such analytical methods as liquid chromatography/mass spectrometry. In the field of proteomics, FFE is a highly versatile technology to support key applications due to prefractionation of samples.
We studied on the effect of various detergents on the pH gradient formed by Ampholine and also on the effect on migration rate of soluble proteins in the presence of detergents.
Nonionic detergents (Emulgen 108, Triton X-100, Brij 35) affected scarcely the pH gradient formation, and the migration rate of proteins was affected but not remarkably. As SDS (anionic detergent) concentration increased, the slope of the pH gradient decreased and the protein peak was transported cathodically.
A new type apparatus was designed for carrier free isoelectric focusing which could be connected to high-performance liquid chromatography. The merits of this apparatus are as follows:
1) The electroosmosis was low due to the plastic quality of electrophoresis tubing instead of the glass tubing.
2) An amount of sample could be increased by increasing the number of tubings for electrophoresis.
3) For fractionation, this apparatus was connected by pumping-out device with peristaltic pump to a UV detector and a fraction collector.
For simplification and quick operation of this apparatus, we studied on basic conditions and on the separation of proteins by isoelectric focusing using carrier free electrophoresis. With incorporation of prefocusing process at high voltage, whole operation time could be shortened to one hour or less.