A continuous band of protein applied to the top of the gel Polyacrylamide gel electrophoresis in transverse migrates electrophoretically in the presence of gradually urea-gradient gels is widely used for the study of increasing concentrations of urea. The resulting pattern changes in conformation and quaternary structure of provides a graphic representation of the changes of proproteins induced by increasing concentrations of urea. tein conformation and quaternary structure that occur However, possible uncertainties regarding the effecupon increasing the urea concentration. However, possitive concentration of urea at any given point across ble uncertainties regarding the effective concentration the gel must be considered when this technique is used of urea at those positions in the gel where the conformafor quantitative purposes. We describe here a simple, tional changes occur must be considered when this techpractical procedure for preparing urea-gradient gels nique is used for quantitative purposes. Measurements discontinuously, by stacking various layers of acrylbased on graphical interpolation between the concentraamide solution with increasing urea concentrations. tions at the extremes of the gradient rely entirely on We show that the urea concentration in different lanes the assumption that the gradient is, in fact, perfectly of discontinuous gradient gels prepared in this way linear. Physical and chemical methods of measuring the can be predetermined accurately enough for quantitaurea concentration across the gel, such as slicing the gel tive purposes and provide as an example the study of across its width after the run, dialyzing the urea out of the dissociation of thyroglobulin in urea. Various leveach slice, and measuring the amount of urea in the els of resolution can be attained by this technique. The dialysate, are time-consuming and impractical, espe-