Bacterial adherence on replicas of sodium dodecyl sulfate-polyacrylamide gels.
Bacterial adherence on replicas of sodium dodecyl sulfate-polyacrylamide gels.
复制标题
十二烷基硫酸钠-聚丙烯酰胺凝胶复制品上的细菌粘附。
DOI：
10.1016/0003-2697(87)90359-9
复制
发表时间：
1987
影响因子：
2.9
通讯作者：
Fisher,SJ
中科院分区：
文献类型：
--
作者：
Prakobphol,A;Murray,PA;Fisher,SJ
设计了一种方法来确定蛋白质复杂混合物中哪些分子可以作为细菌受体发挥作用。唾液蛋白用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离并转移到硝酸纤维素上。用3 H代谢标记或用125 I外部标记的细菌在硝酸纤维素复制品上孵育。在4°C下18小时后，通过重复洗涤复制品除去未结合的细胞，并通过放射自显影术观察放射性标记的细菌结合的条带。通过这种技术，通过受体分子上的碳水化合物残基粘附的具核梭杆菌和识别纤连蛋白的肽部分的金黄色葡萄球菌显示出特异性结合到它们各自的受体。这些结果表明，该方法可用于分析细菌与复杂混合物中存在的分子的碳水化合物或蛋白质部分的结合，例如组成生物液体或组织基质的那些。结构特异性，如某些寡糖形成的识别序列，可以通过向孵育培养基中加入适当的单糖以及寡糖抑制剂来进一步研究。后一种方法是特别重要的，因为大多数糖蛋白携带多个N-和O-连接的碳水化合物取代基，可以作为细菌受体。
A method for determining which molecules in a complex mixture of proteins can function as bacterial receptors was devised. Salivary proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose. Bacteria that were metabolically labeled with3H or externally labeled with125I were incubated on the nitrocellulose replicas. After 18 h at 4°C, the unbound cells were removed by repeated washing of the replicas, and the bands to which the radiolabeled bacteria bound were visualized by autoradiography. By this technique, Fusobacterium nucleatum, which adheres via carbohydrate residues on receptor molecules, and Staphylococcus aureus, which recognizes the peptide portion of fibronectin, were shown to bind specifically to their respective receptors. These results suggest that this method can be useful for profiling bacterial binding to either the carbohydrate or the protein portions of molecules present in complex mixtures, such as those composing biological fluids or tissue substrates. Structural specificities, such as recognition sequences formed by certain oligosaccharides, could be further investigated by adding the appropriate simple sugers, as well as oligosaccharide inhibitors, to the incubation medium. The latter approach is particularly important since most glycoproteins carry multiple N- and O-linked carbohydrate substituents that could serve as bacterial receptors.