Free-flow electrophoresis in a microfabricated chamber with a micromodule fraction separator: Continuous separation of proteins

Continuous free flow electrophoresis of proteins was carried out in a microfabricated free flow electrophoresis (mFFE) module with the 30-μm thick slit of the separation. The newly developed micromodule fraction separator (MFS) was attached to the down-stream end site of the separation chamber of mFFE. By using the MFS, electrolyte solution from the separation chamber was introduced to the peristaltic pump without disturbing the electrolyte solution flow at the bottom side of the chamber. The separation of protein mixture samples was achieved by a hydroxypropylmethylcellulose pretreatment coating of the separation chamber. The pretreatment of the sample chamber effectively suppressed electroosmotic flow. All fractionated samples were collected using the MFS after continuous elecrophoresis and analyzed by reversed-phase HPLC. From the results of HPLC analyses none of the cytochrome c fractions at the other ports revealed cross talk phenomena at adjacent ports. A similar result occurred for the myoglobin. This means that these proteins were completely separated from each other by continuous mFFE, and the MFS functioned efficiently during continuous electrophoresis.