Gel electrophoresis has been used to fractionate macromolecules for studies in all fields of biochemistry and biophysics. To understand mechanisms of sieving during gel electrophoresis, the PIs have previously qualified sieving and tested several models of sieving by gels. Two models, one based on sterics and one and based on hydrodynamics, have survived these tests. During previous studies, sieving for spheres with radii between 5 and 500 nm has been qualified; an analysis of the sieving of rods has been started; the structure of gels has been investigated. The objectives of the present proposal are to: (1) determine the model that most accurately describes the sieving of spheres during gel electrophoresis; video light microscopy (phase contrast and fluorescent) of individual spheres undergoing gel electrophoresis will be performed; (2) determine how sieving depends on the structure of the gel used for electrophoresis; sieving will be quantified by use of procedures of gel electrophoresis previously developed by the PIs; the structure of gels will be determined in part by use of electron microscopy and computer-assisted image processing; (3) determine the effect on sieving of the dimensions and flexibility of rod-shaped particles; for this purpose, one of the PIs' procedures of two-dimensional gel electrophoresis will be used; (4) obtain and organize for rapid access a catalogue of conditions appropriate for sieving-based separation and characterization of particles with effective radii as small as 1- 2 nm and as large as 5-10 nm. The research proposed will contribute to the understanding of motion through porous networks in both biological and non-biological systems. This research will also increase the capacity for rapid and inexpensive biophysical characterization of particles that need not be purified.
