The formation of small-pore gels by an electrically charged agarose derivative

Previous studies have shown that, during the formation of an underivatized agarose gel, agarose molecules laterally aggregate to form thicker fibers called suprafibers; the suprafibers branch to form a gelled network. In the present study, electron microscopy of thin sections is used to investigate both the thickness and the spacing of the fibers of gels formed by agarose chemically derivatized with carboxymethyl (negatively charged) groups. For carboxymethyl agarose, electron microscopy reveals that gels cast in water consist of both fibers narrower and pores smaller than those observed for water-cast underivatized agarose gels at the same concentration. This result is confirmed by using the electrophoretic sieving of spheres to determine the radius (P(E)) of the effective pore of the gel. At a given concentration of gel less than 1%, the P(E) for a water-cast carboxymethyl agarose gel is 0.25-0.30x the P(E) for a water-cast underivatized agarose gel. The value of P(E) predicts the extent of the electrophoretic sieving that is observed when double-stranded DNA is subjected to electrophoresis through a water-cast carboxymethyl agarose gel; DNA bands formed in a water-cast carboxymethyl agarose gel are comparable in quality to DNA bands formed in a water-cast underivatized agarose gel of equal P(E). The following observation supports the hypothesis that electrical charge-charge repulsion among carboxymethyl agarose molecules inhibits the formation of suprafibers in water-cast carboxymethyl agarose gels: Increased content of suprafibers in carboxymethyl agarose gels is observed when the ionic strength is raised by the presence of NaCl, MgCl₂, or any of several buffers during gelation of carboxymethyl agarose.