Quantitative Microdialysis: Experimental Protocol and Software for Small Molecule Protein Affinity Determination and for Exclusion of Compounds with Poor Physicochemical Properties.
Quantitative Microdialysis: Experimental Protocol and Software for Small Molecule Protein Affinity Determination and for Exclusion of Compounds with Poor Physicochemical Properties.
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DOI：
10.3390/mps3030055
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发表时间：
2020-07-30
影响因子：
2.4
通讯作者：
Auer M
中科院分区：
文献类型：
--
作者：
Shave S;Pham NT;Śmieja CB;Auer M
定量微透析是一种传统的生物物理亲和力测定技术。在制定详细的实验方案时，我们使用了市售设备、快速平衡透析 (RED) 装置 (ThermoFisher Scientific)，这意味着它对大多数实验室开放。将目标蛋白和测试化合物在隔开的室中孵育，仅允许小分子转移到更大的储存室，然后使用反相高效液相色谱（RP-HPLC）或液相色谱-质谱（LC-MS）测定每个室中化合物的丰度。在含有目标蛋白的室中测量的较高化合物浓度表明结合。作为一项新颖且独特的贡献，我们提出了一种用于实验数据数学分析的协议。我们提供了方程和软件，以产生高达 0.5 mM KD 的测试化合物-靶蛋白复合物的解离常数，并且我们定量讨论了与测量的化合物浓度相关的亲和力的局限性。
Quantitative microdialysis is a traditional biophysical affinity determination technique. In the development of the detailed experimental protocol presented, we used commercially available equipment, rapid equilibrium dialysis (RED) devices (ThermoFisher Scientific), which means that it is open to most laboratories. The target protein and test compound are incubated in a chamber partitioned to allow only small molecules to transition to a larger reservoir chamber, then reversed-phase high performance liquid chromatography (RP-HPLC) or liquid chromatography–mass spectrometry (LC–MS) is used to determine the abundance of compound in each chamber. A higher compound concentration measured in the chamber that contains the target protein indicates binding. As a novel, and differentiating contribution, we present a protocol for mathematical analysis of experimental data. We provide the equations and the software to yield dissociation constants for the test compound-target protein complex up to 0.5 mM KD, and we quantitatively discuss the limitations of affinities in relation to measured compound concentrations.