Protein-binding assays in biological liquids using microscale thermophoresis
Protein-binding assays in biological liquids using microscale thermophoresis
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DOI：
10.1038/ncomms1093
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发表时间：
2010-10-01
影响因子：
16.6
通讯作者：
Duhr, Stefan
中科院分区：
文献类型：
--
作者：
Wienken, Christoph J.;Baaske, Philipp;Duhr, Stefan
预计人体内的蛋白质相互作用与体外的情况有所不同。这在研究蛋白质功能或开发新药时至关重要。在这项研究中，我们提出了一种样品高效的自由溶液方法，称为微尺度热泳术，能够分析生物液体（如血清或细胞裂解液）中蛋白质或小分子的相互作用。这项技术是基于分子热泳术，它提供了分子大小、电荷和水合壳的信息。我们使用免疫学相关系统验证了该方法，包括人干扰素γ和钙调素与钙的相互作用。在缓冲液和人血清中测定了小分子抑制剂槲皮素对其激酶PKA的亲和力，发现其在血清中的亲和力降低了400倍。这些关于生物基质影响的信息可能使我们对蛋白质的功能得出更可靠的结论，并可能促进更有效的药物开发。
Protein interactions inside the human body are expected to differ from the situation in vitro. This is crucial when investigating protein functions or developing new drugs. In this study, we present a sample-efficient, free-solution method, termed microscale thermophoresis, that is capable of analysing interactions of proteins or small molecules in biological liquids such as blood serum or cell lysate. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge and hydration shell. We validated the method using immunologically relevant systems including human interferon gamma and the interaction of calmodulin with calcium. The affinity of the small-molecule inhibitor quercetin to its kinase PKA was determined in buffer and human serum, revealing a 400-fold reduced affinity in serum. This information about the influence of the biological matrix may allow to make more reliable conclusions on protein functionality, and may facilitate more efficient drug development.