Abstract
Microscale thermophoresis (MST) has become a widely used technique to determine the KD or EC50 of protein–ligand interactions. The method exploits the tendency of macromolecules to migrate along a thermal gradient (i.e., thermophoresis). Differences in thermophoresis as a function of the liganded state of a macromolecule can be measured and assembled into a binding curve that can be analyzed to yield KD. In this protocol, we outline a simple experiment designed for new MST users, with the goal of using readily available, inexpensive materials to plan, execute, and analyze an MST experiment.
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References
Jerabek-Willemsen M, André T, Wanner R et al (2014) MicroScale thermophoresis: interaction analysis and beyond. J Mol Struct 1077:101–113
Duhr S, Braun D (2006) Why molecules move along a temperature gradient. Proc Natl Acad Sci U S A 103:19678–19682
Baaske P, Wienken CJ, Reineck P et al (2010) Optical thermophoresis for quantifying the buffer dependence of aptamer binding. Angew Chem Int Ed 49:2238–2241
Wienken CJ, Baaske P, Rothbauer U et al (2010) Protein-binding assays in biological liquids using microscale thermophoresis. Nat Commun 1:100
Jerabek-Willemsen M, Wienken CJ, Braun D et al (2011) Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol 9:342–353
Lippok S, Seidel SAI, Duhr S et al (2012) Direct detection of antibody concentration and affinity in human serum using microscale thermophoresis. Anal Chem 84:3523–3530
Seidel SAI, Dijkman PM, Lea WA et al (2013) Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59:301–315
Scheuermann TH, Padrick SB, Gardner KH et al (2016) On the acquisition and analysis of microscale thermophoresis data. Anal Biochem 496:79–83
Tso SC, Chen Q, Vishnivetskiy SA et al (2018) Using two-site binding models to analyze microscale thermophoresis data. Anal Biochem 540-541:64–75
Brautigam CA (2015) Calculations and publication-quality illustrations for analytical ultracentrifugation data. Methods Enzymol 562:109–134
Bevington PR, Robinson DK (1992) Data reduction and error analysis for the physical sciences. WCB/McGraw-Hill, Boston, MA
Pace CN, Vajdos F, Fee L et al (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci 4:2411–2423
Fung HYJ, Fu S-C, Brautigam CA et al (2015) Structural determinants of nuclear export signal orientation in binding to exportin CRM1. elife 4:e10034
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Tso, SC., Brautigam, C.A. (2021). Measuring the KD of Protein–Ligand Interactions Using Microscale Thermophoresis. In: Daviter, T., Johnson, C.M., McLaughlin, S.H., Williams, M.A. (eds) Protein-Ligand Interactions. Methods in Molecular Biology, vol 2263. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1197-5_6
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DOI: https://doi.org/10.1007/978-1-0716-1197-5_6
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1196-8
Online ISBN: 978-1-0716-1197-5
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