Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments
Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments
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DOI：
10.1038/nprot.2010.49
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发表时间：
2010-01-01
期刊：
影响因子：
14.8
通讯作者：
Blank, Kerstin
中科院分区：
文献类型：
--
作者：
Zimmermann, Julia L.;Nicolaus, Thomas;Blank, Kerstin
单分子 (SM) 实验的成功关键取决于待研究生物分子的功能固定。随着 SM 荧光与 SM 力实验相结合的持续趋势，需要适用于这两种类型的测量的方法。我们描述了可以用游离硫醇基团制备的任何类型生物分子的位点特异性和共价偶联的通用方案。该方案使用聚乙二醇 (PEG) 间隔基，其一端带有 N-羟基琥珀酰亚胺 (NHS) 基团，另一端带有马来酰亚胺基团。 NHS基团与氨基功能化表面反应后，相对稳定但高反应性的马来酰亚胺基团允许生物分子偶联。该方案提供了具有低荧光背景、低非特异性结合和大量反应位点的表面。 6小时内即可获得含有固定生物分子的表面。
The success of single-molecule (SM) experiments critically depends on the functional immobilization of the biomolecule(s) to be studied. With the continuing trend of combining SM fluorescence with SM force experiments, methods are required that are suitable for both types of measurements. We describe a general protocol for the site-specific and covalent coupling of any type of biomolecule that can be prepared with a free thiol group. the protocol uses a poly(ethylene glycol) (PEG) spacer, which carries an N-hydroxy succinimide (NHS) group on one end and a maleimide group on the other. after reacting the NHS group with an amino-functionalized surface, the relatively stable but highly reactive maleimide group allows the coupling of the biomolecule. this protocol provides surfaces with low fluorescence background, low nonspecific binding and a large number of reactive sites. surfaces containing immobilized biomolecules can be obtained within 6 h.