Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity
Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity
复制标题
测量大分子配体亲和力的等温滴定量热法
DOI：
10.3791/2796
复制
发表时间：
2011-09-01
影响因子：
1.2
通讯作者：
Howell, Elizabeth E.
中科院分区：
文献类型：
--
作者：
Duff, Michael R.;Grubbs, Jordan;Howell, Elizabeth E.
等温滴定量热法（ITC）是了解结合反应的完整热力学图的有用工具。在生物科学中，大分子相互作用对于理解细胞的机制至关重要。实验条件，如缓冲液和温度，可以定制所研究的特定结合系统。然而，需要仔细规划，因为某些配体和大分子浓度范围是获得有用数据所必需的。为了获得可靠的结果，需要准确测定大分子和配体的浓度。在制备样品时也需要小心，因为杂质会显著影响实验。当ITC实验与对照沿着正确进行时，获得有用的结合信息，例如化学计量、亲和力和焓。通过在不同的缓冲液或温度条件下运行额外的实验，可以获得有关系统的更详细的信息。一个ITC实验的基本设置的协议。
Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.