Preparative isolation of protein complexes and other bioparticles by elution from polyacrylamide gels

Abstract

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.

Optimization of a native gel electrophoretic process for the purification of intracellular green fluorescent protein from intact Escherichia coli cells

“…Therefore, purification of GFP using preparative n-PAGE could be an appropriate method for high-throughput separation. However, the cost of expensive devices for preparative gel electrophoresis has always been a factor that leads to its discontinuation as a downstream processing technique [9]. Nevertheless, the technique developed here enables the GFP to be separated and purified electrophoretically from intact cells without disrupting the cells.…”

“…This technique enables GFP to be separated and purified electrophoretically from intact Escherichia coli (E. coli) cells without disrupting the cells. Preparative electrophoresis has been described in numerous publications with many applications (for a review, see [9]). The basic concept of electrophoresis is very simple but the migration of charged particles or ions through a medium is influenced by many factors.…”

Separation of Native Allophycocyanin and R-Phycocyanin from Marine Red Macroalga Polysiphonia urceolata by the Polyacrylamide Gel Electrophoresis Performed in Novel Buffer Systems

“…Among classical discontinuous PAGE modes [29]–[31], the PAGE with Tris−Barbital buffer system is only one that is performed in a near neutral pH buffer system [32]. Even though in such a near neutral pH condition, the R-PC band still showed an observable decrease in its native color with the PAGE performing, implying the R-PCs at least had somewhat dissociation or denaturing during the PAGE.…”

Polyacrylamide Gel Electrophoresis

“…The recovered oligosaccharide also contains oligo-and polyacrylamide, and these contaminating substances are difficult to remove, making it hard to obtain sample of sufficient purity to afford clean nuclear magnetic resonance (NMR) and mass spectral data required for structural assignment. Native continuous elution (CE) PAGE (CE-PAGE) has been used by a number of researchers for the micropreparative separation of proteins, N-glycans, lipopolysaccharides, and nucleic acids [15]. CE-PAGE combines the convenience of a liquid chromatography (LC) system with the many advantages of PAGE, especially the possibility of customizing the experimental conditions for a particular sample mixture without the high additional cost.…”