期刊
ANALYTICAL BIOCHEMISTRY
卷 496, 期 -, 页码 79-93出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2015.12.013
关键词
Microscale thermophoresis; PALMIST; Protein-protein interactions; Isothermal titration calorimetry; Hypoxia-inducible factor; Actin
资金
- Cancer Research and Prevention Institute of Texas [RP 130513]
- Howard Hughes Medical Institute
- NIH [GM56322]
- Welch Foundation [I-1544]
A comprehensive understanding of the molecular mechanisms underpinning cellular functions is dependent on a detailed characterization of the energetics of macromolecular binding, often quantified by the equilibrium dissociation constant, K-D. While many biophysical methods may be used to obtain K-D, the focus of this report is a relatively new method called microscale thermophoresis (MST). In an MST experiment, a capillary tube filled with a solution containing a dye-labeled solute is illuminated with an infrared laser, rapidly creating a temperature gradient. Molecules will migrate along this gradient, causing changes in the observed fluorescence. Because the net migration of the labeled molecules will depend on their liganded state, a binding curve as a function of ligand concentration can be constructed from MST data and analyzed to determine K-D. Herein, simulations demonstrate the limits of K-D that can be measured in current instrumentation. They also show that binding kinetics is a major concern in planning and executing MST experiments. Additionally, studies of two protein protein interactions illustrate challenges encountered in acquiring and analyzing MST data. Combined, these approaches indicate a set of best practices for performing and analyzing MST experiments. Software for rigorous data analysis is also introduced. (c) 2015 Elsevier Inc. All rights reserved.
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