Muñoz G, Marshall S, Cabrera M, Horvat A
Laboratorio de Genética Molecular, Facultad de Ciencias Básicas y Matemáticas, Universidad Católica de Valparaíso, Chile.
Anal Biochem. 1988 May 1;170(2):491-4. doi: 10.1016/0003-2697(88)90663-x.
A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel.
本文描述了一种高灵敏度且简单的方法，用于增强通过一维或二维聚丙烯酰胺凝胶电泳分离的糖蛋白的检测。该方法是对D. Fargeaud等人（D. Fargeaud、J. C. Benoit、F. Kato和G. Chappuis，《病毒学文献》，1984年，第80卷，第69 - 82页）所描述方法的改进，该方法使用与异硫氰酸荧光素偶联的伴刀豆球蛋白A来检测糖蛋白的碳水化合物部分。简要来说，将电泳后的凝胶暴露于荧光凝集素中，充分洗涤，然后依次转移至去离子水中的50%甲醇和无水甲醇中。结果是凝胶突然脱水，变得均匀洁白且坚硬。通过将处理后的凝胶暴露于合适的紫外光源下检测，荧光至少增强了两倍。该方法的灵敏度使我们能够通过其碳水化合物含量检测总蛋白量在0.2微克范围内的纯化免疫球蛋白分子。通过与凝胶硝酸银染色获得的相应多肽图谱进行比较，证明了检测的特异性。
