Gentile F, Veneziani B M, Sellitto C
Centro di Endocrinologia e Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Università Federico II, Naples, Italy.
Anal Biochem. 1997 Jan 15;244(2):228-32. doi: 10.1006/abio.1996.9863.
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.
横向尿素梯度凝胶中的聚丙烯酰胺凝胶电泳被广泛用于研究随着尿素浓度增加而诱导的蛋白质构象和四级结构的变化。然而，当该技术用于定量目的时，必须考虑凝胶上任何给定位置处尿素有效浓度可能存在的不确定性。我们在此描述一种简单实用的方法，通过堆叠不同尿素浓度递增的丙烯酰胺溶液层来间断地制备尿素梯度凝胶。我们表明，以这种方式制备的间断梯度凝胶不同泳道中的尿素浓度可以精确到足以用于定量目的，并以尿素中甲状腺球蛋白的解离研究为例进行了说明。通过该技术可以实现不同程度的分辨率。间断梯度凝胶的使用可能会扩展尿素凝胶电泳在蛋白质构象研究定量方面的用途。
