Polyacrylamide gel electrophoresis in discontinuous transverse urea-gradient gels

Polyacrylamide gel electrophoresis in transverse urea-gradient gels is widely used for the study of changes in conformation and quaternary structure of proteins induced by increasing concentrations of urea. However, possible uncertainties regarding the effective concentration of urea at any given point across the gel must be considered when this technique is used for quantitative purposes. We describe here a simple, practical procedure for preparing urea-gradient gels discontinuously, by stacking various layers of acrylamide solution with increasing urea concentrations. We show that the urea concentration in different lanes of discontinuous gradient gels prepared in this way can be predetermined accurately enough for quantitative purposes and provide as an example the study of the dissociation of thyroglobulin in urea. Various levels of resolution can be attained by this technique. The use of discontinuous gradient gels may extend the usefulness of urea gel electrophoresis in relation with the quantitative aspects of the study of protein conformation. (C) 1997 Academic Press