Righetti P G
Department of Biomedical Sciences and Technologies, University of Milan, Italy.
J Biochem Biophys Methods. 1988 Jun-Jul;16(2-3):99-108. doi: 10.1016/0165-022x(88)90023-1.
The evolution of isoelectric focusing is traced back over the years, from a somewhat shaky origin to present-day immobilized pH gradients. Four generations of methodology are classified and discussed: (A) Kolin's approach, consisting of a two-step technique, generation of a pH gradient by diffusion followed by a rapid electrokinetic protein separation; (B) Svensson-Rilbe's approach, consisting of creating a pH gradient in an electric field by utilizing as buffers a multitude of carrier ampholytes, i.e. of amphoteric species possessing good buffering capacity and conductivity at their pI; (C) immobilized pH gradients, by which non-amphoteric buffers and titrants (acrylamido weak acids and bases), titrated around their pK values, are grafted (insolubilized) onto a polyacrylamide gel matrix and (D) mixed-bed carrier ampholyte-Immobiline gel, by which a soluble, carrier ampholyte generated pH gradient coexists in the same matrix with an insoluble, Immobiline generated, pH gradient.
等电聚焦的发展历程历经多年，从一个略显不稳定的起源发展到如今的固定化pH梯度。本文对四代方法进行了分类和讨论：（A）科林方法，包括两步技术，通过扩散产生pH梯度，随后进行快速电动蛋白质分离；（B）斯文森-里尔贝方法，通过使用多种载体两性电解质作为缓冲剂在电场中创建pH梯度，即具有良好缓冲能力且在其pI处具有导电性的两性物质；（C）固定化pH梯度，通过将非两性缓冲剂和滴定剂（丙烯酰胺弱酸和弱碱）在其pK值附近滴定后接枝（不溶解）到聚丙烯酰胺凝胶基质上；（D）混合床载体两性电解质-固定化凝胶，通过该方法，可溶性载体两性电解质产生的pH梯度与不溶性固定化凝胶产生的pH梯度共存于同一基质中。
