Surface passivation for single-molecule protein studies.
Surface passivation for single-molecule protein studies.
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DOI：
10.3791/50549
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发表时间：
2014-04-24
期刊：
影响因子：
--
通讯作者：
Joo C
中科院分区：
文献类型：
--
作者：
Chandradoss SD;Haagsma AC;Lee YK;Hwang JH;Nam JM;Joo C
单分子荧光光谱学已被证明有助于在纳米尺度上理解广泛的生物现象。这项技术能够为生物科学带来什么的重要例子是对蛋白质-蛋白质和蛋白质-核酸相互作用的机械洞察力。当在单分子水平上探测蛋白质之间的相互作用时，蛋白质或其底物往往固定在玻璃表面上，这使得可以进行长期的观察。这种固定方案可能会引入不需要的表面伪影。因此，必须对玻璃表面进行钝化，使其成为惰性的。使用聚乙二醇(PEG)的表面涂层因其在防止蛋白质与玻璃表面非特异性相互作用方面的高性能而脱颖而出。然而，聚合物涂层过程是困难的，因为一系列表面处理引起的复杂性，以及在过程结束时表面需要没有任何荧光分子的严格要求。在这里，我们提供了一个健壮的协议和分步说明。它包括表面清洁，包括食人鱼蚀刻，表面官能化与胺基团，最后聚乙二醇涂层。为了获得高密度的聚乙二醇层，我们引入了一种新的策略，用聚乙二醇分子分两轮处理表面，显著提高了钝化质量。我们为每个关键步骤提供了具有代表性的结果以及实用的建议，以便任何人都能实现高质量的表面钝化。
Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.
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影响因子：
3.3
作者：
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发表时间：
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期刊：
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