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Journal article
On the acquisition and analysis of microscale thermophoresis data
Analytical biochemistry, v 496
01 Mar 2016
PMID: 26739938
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
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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Details
- Title
- On the acquisition and analysis of microscale thermophoresis data
- Creators
- Thomas H. Scheuermann - The University of Texas Southwestern Medical CenterShae B. Padrick - The University of Texas Southwestern Medical CenterKevin H. Gardner - CUNY Advanced Science Research CenterChad A. Brautigam - The University of Texas Southwestern Medical Center
- Publication Details
- Analytical biochemistry, v 496
- Publisher
- Elsevier
- Number of pages
- 15
- Grant note
- I-1544 / Welch Foundation; The Welch Foundation Howard Hughes Medical Institute RP 130513 / Cancer Research and Prevention Institute of Texas; Cancer Prevention & Research Institute of Texas GM56322 / NIH; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Biochemistry and Molecular Biology
- Web of Science ID
- WOS:000370304100014
- Scopus ID
- 2-s2.0-84955247395
- Other Identifier
- 991020836477904721
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Data related to this publication, from InCites Benchmarking & Analytics tool:
- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemical Research Methods
- Biochemistry & Molecular Biology
- Chemistry, Analytical