Free-flow electrophoresis
The free-flow electrophoresis (also tragerfreie electrophoresis or free-flow electrophoresis called ) is a continuous ( semi) preparative electrophoretic separation method of the biochemistry that is used in particular for obtaining purified proteins, protein complexes, peptides and organelles in larger amounts, as for preparative method must be available.
Properties
Typical applications for the free-flow electrophoresis (FFE ), the separation of complex protein mixtures, and isolation of protein isoforms, the purification of particles, organelles or cells for further analysis. An advantage of the FFE method is that the separation is carried out very quickly and gently in an aqueous medium and no interaction with a solid matrix, such as polyacrylamide gel electrophoresis in takes place. Thus, the recovery rate is relatively high, since no theoretical analyte lost. The FFE separations always run off continuously, thus it is possible to purify large amounts of the analytes. In commercial systems, the throughput is between one to four milligrams of the sample per hour. Furthermore, the separations of proteins can be carried out both native and denaturing.
Method
A uniform laminar flow of fluid is passed through the two plates, wherein it is arranged on the end of the plates in parallel, similar capillary tube and then flows into collecting vessels, microtiter plates, usually is conducted.
Transverse to the flow direction is applied an electric voltage to the buffered solution. When a protein solution is introduced to the sample application site, the proteins are separated during transport through the separation chamber according to the charge density or the isoelectric point so that they end up in different capillary tubes. With appropriate standardization and control of all parameters, the process is a continuous method for the preparation of large quantities of differently charged molecules.
There can be different electrophoretic methods in the separation chamber to apply:
- Isoelectric focusing, separation takes place according to the isoelectric point
- Zone electrophoresis separation according to charge density
- Isotachophoresis, separation by electrophoretic mobility
History
The Free Flow Electrophoresis method was developed mainly in the 1960s by Kurt Hannig at the Max Planck Institute of Biochemistry. It was until the 1980s a standardized method for separating cells and organelles. In addition, numerous Free Flow Electrophoresis experiments were performed in the Spacelab, to minimize the influence of sedimentation in gravity-free space. The introduction of flow cytometry finished part, the application of FFE for cell separation and the applications of the Free Flow Electrophoresis shifted largely to date on the separation of particles, proteins and peptide mixtures. Several research groups are also working on miniaturized FFE systems ( μFFE ) for various purposes.
Structure of the FreeFlow electrophoresis
The separation chamber is ever of a back plate and a front plate. The back panel consists of a coolable aluminum block, which is coated with a kuststoffbeschichteten and mirrored glass sheet. The front plate is usually made of acrylic glass (PMMA). The distance between the plates is usually used in commercial systems from 0.1 to 0.5 mm. In the front panel there are the inlets for the separation media, the sample inlets, the Fraktionierschläuche and the electrodes. The separation media and samples are each supported via a peristaltic pump to the separation chamber. It is important that a laminar flow is present. Perpendicular to the flow direction of the electric field is established across the electrodes. At the end of the separating chamber, the separated sample is taken in 96 Fraktionierschläuchen and passed into the collecting vessels.