Structure of agarose gels: Mean pore radius

Abstract

To use an agarose gel's sieving for the biophysical characterization of macromolecules and supramolecular complexes, the effective gel's pore radius (PE) is determined as a function of the agarose percentage (A). In previous studies, performed by use of agarose gel electrophoresis, the sieving of spheres of known radius (R) was used, together with two different (unproven) theories of sieving, to obtain PE. The PE values obtained were self-consistent and independent of R. The PE vs. A relationship did not agree with that predicted by a model that represents the gel as straight, randomly-oriented fibers (random fiber model). To test the accuracy of both the empirical PE vs. A relationship and the previously assumed model of gel structure, in the present study thin sections of agarose gels have been examined by use of electron microscopy.Gels of the agarose previously used to quantify sieving (SeaKem LE, Marine Colloids) were cast in the buffer previously used: 0.025 M sodium phosphate, pH 7.4, 0.001M MgCl2. Pieces of gel were fixed with osmium tetroxide, dehydrated and embedded in Epon. Dark gold (120 nm) sections were examined with a JEM-100CX transmission electron microscope. Electron micrographs were captured with a DataTranslation QuickCapture video digitizer and were processed on a Macintosh II computer using the program, Image.4