Masson Patrick, Pashirova Tatiana
Institute of Fundamental Medicine and Biology, Kazan Federal University, 18 Kremlyovskaya St., 420008 Kazan, Russia.
Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center of RAS, Arbuzov Str. 8, 420088 Kazan, Russia.
Int J Mol Sci. 2025 Apr 5;26(7):3409. doi: 10.3390/ijms26073409.
Affinity gel electrophoresis was introduced about 50 years ago. Proteins interact with a ligand immobilized in the support. Specific interactions cause a decrease in electrophoretic mobility. The presence of a free ligand, competing with an immobilized ligand, restores electrophoretic mobility. In affinity capillary electrophoresis, the ligand is mobile, and its interaction with a specific protein changes the mobility of the protein-ligand complex. This review mostly focuses on gel affinity electrophoresis. The theoretical basis of this technique, ligand immobilization strategies, and principles for determination of ligand affinity are addressed. Factors affecting specificity and strength of interactions are discussed, in particular, the structure of the affinity matrix, pH, temperature, hydrostatic pressure, solvent, co-solvents, electric field, and other physico-chemical conditions. Capillary affinity electrophoresis principles and uses are also briefly introduced. Affinity gel electrophoresis can be used for qualitative and quantitative purposes. This includes detection of specific proteins in complex media, investigation of specific interactions, protein heterogeneity, molecular and genetic polymorphism, estimation of dissociation constants of protein-ligand complexes, and conformational stability of binding sites. Future prospects, in particular for screening of engineered mutants and potential new drugs, coupling to other analytical methods, and ultra-microtechnological developments, are addressed in light of trends and renewal of this old technique.
亲和凝胶电泳大约在50年前被引入。蛋白质与固定在支持物上的配体相互作用。特异性相互作用导致电泳迁移率降低。游离配体与固定化配体竞争，可恢复电泳迁移率。在亲和毛细管电泳中，配体是可移动的，它与特定蛋白质的相互作用会改变蛋白质-配体复合物的迁移率。本综述主要关注凝胶亲和电泳。阐述了该技术的理论基础、配体固定化策略以及配体亲和力测定原理。讨论了影响相互作用特异性和强度的因素，特别是亲和基质的结构、pH值、温度、静水压力、溶剂、共溶剂、电场和其他物理化学条件。还简要介绍了毛细管亲和电泳原理及应用。亲和凝胶电泳可用于定性和定量目的。这包括检测复杂介质中的特定蛋白质、研究特异性相互作用、蛋白质异质性、分子和遗传多态性、估计蛋白质-配体复合物的解离常数以及结合位点的构象稳定性。鉴于这项古老技术的发展趋势和更新情况，探讨了其未来前景，特别是在工程突变体和潜在新药筛选、与其他分析方法联用以及超微技术发展方面的前景。
