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Author:
Date:
2016-03-05
[Abstract] The displacement assay was designed to quantify the direct competition between two homologous ribosomal proteins from Mycobacterium tuberculosis, S18-1 and S18-2, for interaction with their cognate binding partner, ribosomal protein S6 (Prisic et al., 2015). The S18 proteins were dialyzed in two physiologically relevant conditions (i.e. in the presence of Zn2+ or with EDTA to chelate Zn2+) and then allowed to compete for binding to S6 which was maintained in limiting concentration. The result was obtained through an ELISA, where S6-His is first bound to a Ni2+-NTA plate, followed by addition of S18-2 in excess to S6, then by addition of increasing concentrations of S18-1. The percentage of S18-2 that remained bound to S6 was ...
[摘要] 设计置换测定以定量来自结核分枝杆菌（Mycobacterium tuberculosis）S18-1和S18-2的两个同源核糖体蛋白质之间的直接竞争，用于与它们的同源结合伴侣，核糖体蛋白S6（Prisic et al。，2015）。 S18蛋白质在两种生理相关条件下（即在Zn 2+ 2+存在下或用EDTA螯合Zn 2+ 2+）进行透析，并且然后允许竞争结合S6，其维持在有限浓度。通过ELISA获得结果，其中S6-His首先结合Ni 2+ 2+ -NTA板，然后加入超过S6的S18-2，然后加入递增浓度的S18-1。在化学发光ELISA中用S18-2蛋白和第二抗体特异性的抗体定量保留结合S6的S18-2的百分比。以这种方式，S18-1蛋白质被S18-1蛋白质的置换报告为通过用S18-2饱和S6实现的全强度信号的百分比。在其基础上，该方法利用天然蛋白质 - 蛋白质相互作用，并且可以应用于其中两种或更多种蛋白质竞争结合上述靶配体的其他系统。
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