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Gel electrophoresis and size exclusion chromatography (SEC) are vital techniques in biochemical research, employing gel matrix structures made of polysaccharides or synthetic polymers like polyacrylamide for the analysis and separation of macromolecules. Polysaccharides, such as agarose, offer safer alternatives to acrylamide. Polysaccharide gels, notably agarose, facilitate the analysis and purification of proteins and nucleic acids through a molecular sieving mechanism. Gel electrophoresis for proteins is mainly divided into denaturing and native methods. Denaturing electrophoresis with sodium dodecyl sulfate (SDS) simplifies protein migration but disrupts molecular interactions. Conversely, native gel electrophoresis, without SDS, allows proteins to migrate based on the running pH and the isoelectric point of the proteins, while nucleic acids consistently migrate toward the anode. The electrophoresis of proteins with variable charges presents complexes. This review focuses on the use of polysaccharides, particularly agarose, for native gel electrophoresis, highlighting their applications in separating macromolecules. It also discusses the applications and limitations of agarose gels when used as a matrix for electrophoresis. Such information should help in designing electrophoresis experiments using polysaccharides.

We have developed a new Western blotting method of native proteins from agarose-based gel electrophoresis using a buffer at pH 6.1 containing basic histidine and acidic 2-(N-morpholino)ethanesulfonic acid. This gel electrophoresis successfully provided native structures for a variety of proteins and macromolecular complexes. This paper is focused on the Western blotting of native protein bands separated on agarose gels. Two blotting methods from agarose gel to PVDF membrane are introduced here, one by contact (diffusion) blotting and another by electroblotting after pre-treating the agarose gels with SDS. The contact blotting resulted in the transfer of native GFP, native human plexin domain containing protein 2 (PLXDC2) and native SARS-CoV-2 spike protein, which were detected by conformation-specific antibodies generated in-house.

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography.

In this study, we review the agarose native gel electrophoresis that separates proteins and macromolecular complexes in their native state and transfer of the separated proteins from the agarose gel to membranes by contact blotting which retains the native state of these structures. Green fluorescent protein showed functional state both on agarose gel and blotted membrane. Based on the combined procedures, we discovered conformation-specific monoclonal antibodies against PLXDC2 and SARS-CoV-2 spike protein.