Free-flow electrophoresis


Free-flow electrophoresis , also known as carrier-free electrophoresis, is a matrix-free electrophoretic separation technique. FFE is an analogous technique to capillary electrophoresis, with a comparable resolution, that can used for scientific questions, where semi-preparative and preparative amounts of samples are needed. It is used to quantitatively separate samples according to differences in charge or isoelectric point. Because of the versatility of the technique, a wide range of protocols for the separation of samples like rare metal ions, protein isoforms, multiprotein complexes, peptides, organelles, cells, DNA origami, blood serum and nanoparticles exist. The advantage of FFE is the fast and gentle separation of samples dissolved in a liquid solvent without any need of a matrix, like polyacrylamide in gel electrophoresis. This ensures a very high recovery rate since analytes do not adhere to any carrier or matrix structure. Because of its continuous nature and high volume throughput, this technique allows a fast separation of preparative amounts of samples with a very high resolution. Furthermore, the separations can be conducted under native or denaturing conditions.

History

FFE was developed in the 1960s by Kurt Hannig at the Max-Planck-Institute in Germany. Until the 1980s, it was a standardized technology for the separation of cells and organelles, and FFE was even tested in space to minimize the sedimentation under zero gravity. As flow cytometry became the standard method for cell sorting, FFE developments focused on the separation of proteins and charged particles. Some groups are also working on miniaturized versions of FFE systems or micro FFEs.

Technique

The separation chamber consists of a backplate and a front plate. The backplate usually consists of a cooled aluminum block, covered with a plastic covered glass mirror. The front plate is nowadays made of PMMA, in earlier times glass has been used. The distance between the front- and the backplate can be adjusted by spacers and is usually between 0.1 - 0.5 mm. The front plate also contains the inlets for the separation buffers and the sample, the outlets for the fractionation tubes and the platinum wires as electrodes. By applying different buffers over the multiple buffer inlets the operator is able to change pH, salt concentrations or composition and therefore the separation conditions over the width of the chamber.
The separation buffers and the sample are applied by peristaltic pumps, to ensure a laminar flow. A high voltage electric field is applied perpendicular to the laminar flow. Analytes in the laminar flow can be separated by charge density and/or isoelectric point. Because of its highly versatile nature, this technique can make use of different modes of electrophoresis, like for example isotachophoresis, isoelectric focusing or zone electrophoresis.
At the end of the separation cell, the separated sample is split up at the fractionation tubes and collected in microtiter plates. Afterwards the samples can be characterized by all major techniques like HPLC, LC-MS, mass spectrometry or electrophoresis.

Applications

Standard application include the high-resolution separation of protein complexes, membrane proteins, protein and antibody isoforms, cells, subcellular compartments and liposomes. By making use of very flat pH-gradients, generated by ampholytes, it is possible to separate protein isoforms, that vary by less than 0.02 pH units, with a throughput of around 3 mg/h.
It is also possible to add additives to the buffers, to increase the resolution or denature the samples. Typically concentrations of urea up to 8M, 0.1-1% of detergents like: CHAPS, CHAPSO, Digitonin, Dodecyl-ß-D-maltoside, Octyl-ß-D-glucoside, Triton-X-114 and DTT up to 50 mM are tolerated.

Key features