# Gel Shrinkage in Discontinuous Electrophoresis: How to Stabilize the Electrolyte Boundary in EpitachophoresisPart 1Gel Selection

## TL;DR

This paper studies how to prevent gel shrinkage in a special type of electrophoresis called epitachophoresis, focusing on gel selection and stabilization for efficient DNA concentration.

## Contribution

The study introduces optimized agarose gel formulations and conditions to stabilize electrolyte boundaries and maximize DNA recovery in large-volume epitachophoresis.

## Key findings

- Agarose gels with low electroosmotic flow and optimized conditions achieved up to 100% DNA recovery.
- Polyacrylamide gels showed mechanical stability but hindered large DNA fragment concentration due to sieving effects.
- pH gradients at the electrolyte interface were identified as a key factor in gel shrinkage.

## Abstract

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.