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 s tructure of proteins induced by increasing concentrations of urea. How ever, possible uncertainties regarding the effective concentration of urea at any given point across the gel must be considered when this te chnique 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 lane s of discontinuous gradient gels prepared in this way can be predeterm ined accurately enough for quantitative purposes and provide as an exa mple the study of the dissociation of thyroglobulin in urea. Various l evels of resolution can be attained by this technique. The use of disc ontinuous gradient gels may extend the usefulness of urea gel electrop horesis in relation with the quantitative aspects of the study of prot ein conformation. (C) 1997 Academic Press