Regeneration of PEG slide for multiple rounds of single-molecule measurements
Regeneration of PEG slide for multiple rounds of single-molecule measurements
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DOI：
10.1016/j.bpj.2021.02.031
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发表时间：
2021-05-04
影响因子：
3.4
通讯作者：
Myong，Sua
中科院分区：
文献类型：
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作者：
Paul，Tapas;Ha，Taekjip;Myong，Sua
蛋白质和其他生物分子的单分子荧光检测需要聚乙二醇（PEG）钝化的表面。PEG钝化载玻片上的单个通道通常仅使用几次，限制了每个载玻片的实验数量。在这里，我们报告了几种策略，用于再生PEG表面的多轮实验。首先，我们通过0.1%十二烷基硫酸钠洗脱结合的蛋白质来再生DNA或RNA束缚的表面，这比6 M尿素，6 M GdmCl或100μM蛋白酶K更有效。引人注目的是，在五个不同的系统中进行的10次连续实验在分子计数和蛋白质活性方面都产生了难以区分的结果。第二，解旋酶解旋或50 mM NaOH变性的DNA与互补链再退火，以再生具有极高回收率的解旋底物。第三，通过使用7 M NaOH使生物素-PEG层再生以剥离NeutrAvidin，其可重新应用于另外的实验。我们展示了五个循环的再生抗体固定的表面，通过三种不同的蛋白质活性进行了测量。总之，我们的方法代表了可靠和可重复的，但简单和快速的策略，将提高单分子实验的效率。
Single-molecule fluorescence detection of protein and other biomolecules requires a polyethylene glycol (PEG)-passivated surface. Individual channels on a PEG-passivated slide are typically used only a few times, limiting the number of experiments per slide. Here, we report several strategies for regenerating PEG surfaces for multiple rounds of experiments. First, we show regeneration of DNA- or RNA-tethered surfaces by washing out the bound protein by 0.1% sodium dodecyl sulfate, which is significantly more effective than 6 M urea, 6 M GdmCl, or 100μM proteinase K. Strikingly, 10 consecutive experiments in five different systems produced indistinguishable results both in molecule count and protein activity. Second, duplexed DNA unwound by helicase or denatured by 50 mM NaOH was reannealed with a complementary strand to regenerate the duplexed substrate with an exceptionally high recovery rate. Third, the biotin-PEG layer was regenerated by using 7 M NaOH to strip off NeutrAvidin, which can be reapplied for additional experiments. We demonstrate five cycles of regenerating antibody immobilized surface by which three different protein activity was measured. Altogether, our methods represent reliable and reproducible yet simple and rapid strategies that will enhance the efficiency of single-molecule experiments.