Free-flow electrophoresis

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The free-flow electrophoresis (including carrier-free electrophoresis, or free-flow electrophoresis called) is a continuous (semi) preparative electrophoretic separation method of Biochemistry , the purified particular for obtaining proteins , protein complexes , peptides , DNA origami and organelles is used in larger amounts, such as they must be available for preparative procedures.

properties

Typical applications for free flow electrophoresis (FFE) are the separation of complex protein mixtures, isolation of protein isoforms and the purification of particles, organelles or cells for further analysis. One advantage of the FFE method is that the separation takes place very quickly and gently in an aqueous medium and there is no interaction with a solid matrix, such as polyacrylamide in gel electrophoresis . The recovery rate is thus comparatively high, since theoretically no analytes are lost. The FFE separations are always continuous, making it possible to purify large amounts of the analytes. In commercial systems, the throughput is between one to four milligrams of sample per hour. Furthermore, the separations for proteins can be carried out both with preservation of the original conformation and denaturing .

Procedure

A uniform, laminar flow of liquid is passed through two plates, flowing into capillary tubes of the same type arranged in parallel at the end of the plates, and from there into collecting vessels, usually microtiter plates .

Schematic mode of operation of free flow electrophoresis

An electrical voltage is applied to the buffered solution across the direction of flow. If a protein solution is introduced at the sample application point, 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 suitable standardization and control of all parameters, the process is suitable as a continuous method for the preparation of larger quantities of differently charged molecules .

Various electrophoretic processes can be used in the separation chamber:

history

The free flow electrophoresis process was largely developed in the 1960s by Kurt Hannig at the Max Planck Institute for Biochemistry . It was a standardized separation process for cells and organelles until the 1980s. In addition, numerous free flow electrophoresis tests were carried out in Spacelab in order to minimize the influence of sedimentation in the gravitation-free space. The introduction of flow cytometry partially ended the use of FFE for cell separation and the applications of free flow electrophoresis have largely shifted to the separation of particles, proteins and peptide mixtures. Some research groups are also working on miniaturized FFE systems (µFFE) for various purposes.

Construction of the free flow electrophoresis apparatus

Construction of a free flow electrophoresis instrument

The separation chamber consists of a back plate and a front plate. The back plate consists of a coolable aluminum block, which is coated with a plastic-coated and mirrored glass plate. The front panel usually consists of acrylic glass (PMMA) . In commercial systems, the distance between the plates is usually 0.1-0.5 mm. The inlets for the separation media, the sample inlets, the fractionation tubes and the electrodes are located in the front panel. The separation media and samples are each conveyed into the separation chamber via a hose pump. It is important that there is a laminar flow . The electric field is built up across the electrodes perpendicular to the direction of flow . At the end of the separation chamber, the separated sample is tapped in 96 fractionation tubes and directed into the collecting vessels.

Web links

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  1. ↑ Free Flow Elektrophoresis, Kurt Hannig and Hans G. Heidrich, GIT Verlag 1990, ISBN 3-921956-88-9
  2. ↑ Electrophoresis Operations in Space (PDF; 4.6 MB)
  3. ↑ Free-flow electrophoresis in the proteomic era: a technique in flux. Islinger M, Eckerskorn C, Volkl A. Electrophoresis. 2010 Jun; 31 (11): 1754-63. doi: 10.1002 / elps.200900771 . Review. PMID 20506416
  4. ↑ S. Jezierski, L. Gitlin, S. Nagl, D. Belder Multistep liquid-phase lithography for fast prototyping of microfluidic free-flow-electrophoresis chips, Anal. Bioanal. Chem., 2011, 401, 2651-2656. PMID 21892629
  5. ↑ Miniaturizing free-flow electrophoresis - a critical review. Kohlheyer D, Eijkel JC, van den Berg A, Schasfoort RB. Electrophoresis. 2008 Mar; 29 (5): 977-93. doi: 10.1002 / elps.200700725 . Review. PMID 18232029
  6. ↑ Principle of the FFE separations