微尺度热电泳(MST)是一种用于测量分子相互作用强度的技术。MST是基于热泳学的技术,其响应于由IR激光触发的温度梯度而监测与荧光标记的分子的运动相关的荧光变化。与其他检查分子相互作用的方法相比,MST具有优势,如等温滴定量热法,核磁共振,生物层干涉法,和表面等离子体共振,需要一个小的样品大小,不需要固定和高灵敏度的荧光检测。此外,由于该方法涉及将样品加载到易于密封的毛细管中,它可以适用于分析氧敏感样品。在这个生物协议中,我们描述了我们为使用MST检查蛋白质-蛋白质相互作用所做的故障排除和优化,蛋白质-配体相互作用,和蛋白质-纳米晶体相互作用。所开发程序中的重要元素包括1)在厌氧室中的加载和密封能力,以使用位于空气中的台上的NanoTemperMST进行分析,2)识别与数据采集兼容的最佳还原剂,并有效防止痕量氧,3)数据采集和分析程序的优化。该程序为定义这些技术要求苛刻的系统中分子相互作用的决定因素奠定了基础。主要特征•建立了在厌氧室中装载和密封管以进行后续分析的程序。•连二亚硫酸钠(NaDT)可以很容易地用一个电子还原的1,1'-双(3-磺酸丙基)-4,4'-联吡啶鎓[(SPr)2V•]代替,以对氧敏感蛋白进行灵敏的生物物理测定。MoFe蛋白。•建立了MST作为实验工具,以量化对氧极其敏感的新型酶-量子点生物杂交复合物中的结合亲和力。
Microscale thermophoresis (MST) is a technique used to measure the strength of molecular interactions. MST is a thermophoretic-based technique that monitors the change in fluorescence associated with the movement of fluorescent-labeled molecules in response to a temperature gradient triggered by an IR LASER. MST has advantages over other approaches for examining molecular interactions, such as isothermal titration calorimetry, nuclear magnetic resonance, biolayer interferometry, and surface plasmon resonance, requiring a small sample size that does not need to be immobilized and a high-sensitivity fluorescence detection. In addition, since the approach involves the loading of samples into capillaries that can be easily sealed, it can be adapted to analyze oxygen-sensitive samples. In this Bio-protocol, we describe the troubleshooting and optimization we have done to enable the use of MST to examine protein-protein interactions, protein-ligand interactions, and protein-nanocrystal interactions. The salient elements in the developed procedures include 1) loading and sealing capabilities in an anaerobic chamber for analysis using a NanoTemper MST located on the benchtop in air, 2) identification of the optimal reducing agents compatible with data acquisition with effective protection against trace oxygen, and 3) the optimization of data acquisition and analysis procedures. The procedures lay the groundwork to define the determinants of molecular interactions in these technically demanding systems. Key features • Established procedures for loading and sealing tubes in an anaerobic chamber for subsequent analysis. • Sodium dithionite (NaDT) could easily be substituted with one electron-reduced 1,1\'-bis(3-sulfonatopropyl)-4,4\'-bipyridinium [(SPr)2V•] to perform sensitive biophysical assays on oxygen-sensitive proteins like the MoFe protein. • Established MST as an experimental tool to quantify binding affinities in novel enzyme-quantum dot biohybrid complexes that are extremely oxygen-sensitive.