Peter Weber

Isolation of Organelles and Prefractionation of Protein Extracts Using Free‐Flow Electrophoresis

One of the major obstacles in the analysis of proteomes is the extreme complexity of any particular cell or biological fluid. Free‐flow electrophoresis (FFE) is a powerful tool for reduction of this complexity, which is a prerequisite for systematic and comprehensive protein analyses. Protocols are provided in this unit for sample fractionation at two different stages: on the protein level by isoelectric focusing FFE fractionation of crude protein mixtures such as whole cell lysates, and on a subcellular level by zone‐electrophoretic FFE purification of organelles.

Free-Flow Electrophoresis

Publisher Summary Free-flow isoetectric focusing of protein mixtures is one of the methods that fulfill the prerequisites to meet the prefractionation demands to increase the amount of low abundant proteins and to dramatically reduce the complexity of protein mixtures. The power and resolution of free-flow isoelectric focusing are illustrated with the analysis of pig serum using a Pro Team free-flow electrophoresis (FFE) instrument. One needs to reduce the force of all separation chamber clamps, open the clamps pairwise from the outside to the inside, and open the separation chamber by carefully pulling the front part. Place membranes on electrodes starting from the top to the bottom. The smooth side of the membrane should face toward the electrode seal. The membrane must not protrude over the electrode seal. Subsequently, place the paper strips congruently on the membranes in the same fashion. Place all the media and counterblow tubes in a bottle with distilled water. Place the sample tube in an empty 2-ml reaction tube without tightening the screw.