Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels.
Due to its unmatched resolution, gel electrophoresis is an indispensable tool for the analysis of diverse biomolecules. By adaptation of the electrophoretic conditions, even fragile protein complexes as parts of intracellular networks migrate through the gel matrix under sustainment of their integrity. If the thickness of such native gels is significantly increased compared to the analytical version, also high sample loads can be processed. However, the cage-like network obstructs an in-depth analysis for deciphering structure and function of protein complexes and other species. Consequently, the biomolecules have to be removed from the gel matrix into solution. Several approaches summarized in this review tackle this problem. While passive elution relies on diffusion processes, electroelution employs an electric field to force biomolecules out of the gel. An alternative procedure requires a special electrophoresis setup, the continuous elution device. In this apparatus, molecules migrate in the electric field until they leave the gel and were collected in a buffer stream. Successful isolation of diverse protein complexes like photosystems, ATP-dependent enzymes or active respiratory supercomplexes and some other bioparticles demonstrates the versatility of preparative electrophoresis. After liberating particles out of the gel cage, numerous applications are feasible. They include elucidation of the individual components up to high resolution structures of protein complexes. Therefore, preparative electrophoresis can complement standard purification methods and is in some cases superior to them.
Seelert
Holger
H
Department of Chemistry, Physical Biochemistry, Technische Universität Darmstadt, Darmstadt, Germany. seelert@hrzpub.tu-darmstadt.de
Krause
Frank
F
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D046912:Multiprotein Complexes / Q000302:isolation & purification*
D046912:Multiprotein Complexes