Gel Shrinkage in Discontinuous Electrophoresis: How to Stabilize the Electrolyte Boundary in EpitachophoresisPart...

Gel electrophoresis is typically performed in a single electrolyte system. During the development of epitachophoresis for large-volume DNA concentration, which employs a discontinuous electrolyte system, we found that some gels tend to shrink significantly as the boundary between the leading electrolyte (LE) and trailing electrolyte (TE) moves along the gel. Effective stabilization of this boundary is crucial for analyte focusing, particularly in systems processing tens of milliliters of a sample. This study systematically evaluated various gel stabilization mediaincluding agarose-based gels (NEEO (no electroendosmosis), IsoGel, pulsed-field electrophoresis gel) and polyacrylamide gelsbased on their ability to maintain a stable LE/TE boundary, minimize gel shrinkage, and maximize DNA recovery. Agarose gels with low electroosmotic flow were optimized by adjusting the gel concentration, electrolyte composition, and pH and by incorporating Ba²⁺ ions to reduce gel deformation caused by thermal and electrokinetic effects. Computer simulations highlighted pH gradients at the LE/TE interface as a key factor contributing to gel shrinkage. The study also revealed that careful control of the buffer composition and pH, especially when Tris, Bis-Tris, and Bis-Tris propane counterions are used, is essential for stable separation and reproducible DNA recovery. Optimal conditions for agarose gels yielded up to 100% DNA recovery, as confirmed by fluorescence-based quantification and capillary electrophoresis. Polyacrylamide gel demonstrated mechanical stability without shrinkage; however, significant sieving effects hindered the effective concentration of large DNA fragments, limiting its applicability. Overall, agarose gels designed for pulsed-field electrophoresis and optimized NEEO agarose formulations provided the best balance of stability, low analyte interaction, and high recovery efficiency for epitachophoretic DNA concentration. This work summarizes practical approaches to LE/TE interface stabilization, which is critical for large-scale biomolecular separations by epitachophoresis.